Substituted indol-5-ol derivatives and their therapeutical applications

ABSTRACT

The present invention relates generally to the use of compounds to treat a variety of disorders, diseases and pathologic conditions and more specifically to the use of substituted indol-5-ol derivatives to modulate protein kinases and for treating protein kinase-mediated diseases.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplications 61/722,537 (Filed on Nov. 5, 2012) and 61/852,309 (Filed onMar. 15, 2013), both of which are hereby incorporated by reference intheir entirities.

FIELD OF THE INVENTION

The present invention relates generally to the use of compounds to treata variety of disorders, diseases and pathologic conditions and morespecifically to the use of substituted indol-5-ol derivatives tomodulate protein kinases and for treating protein kinase-mediateddiseases.

BACKGROUND OF THE INVENTION

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell. Protein kinases, containing asimilar 250-300 amino acid catalytic domain, catalyze thephosphorylation of target protein substrates.

The kinases may be categorized into families by the substrates in thephosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids,etc.). Tyrosine phosphorylation is a central event in the regulation ofa variety of biological processes such as cell proliferation, migration,differentiation and survival. Several families of receptor andnon-receptor tyrosine kinases control these events by catalyzing thetransfer of phosphate from ATP to a tyrosine residue of specific cellprotein targets. Sequence motifs have been identified that generallycorrespond to each of these kinase families [Hanks et al., FASEB J.,(1995), 9, 576-596; Knighton et al., Science, (1991), 253, 407-414;Garcia-Bustos et al., EMBO J., (1994), 13:2352-2361). Examples ofkinases in the protein kinase family include, without limitation, abl,Akt, bcr-abl, Blk, Brk, Btk, c-kit, c-Met, c-src, c-fms, CDK1, CDK2,CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, cRaf1, CSF1R, CSK,EGFR, ErbB2, ErbB3, ErbB4, Erk, Fak, fes, FGFR1, FGFR2, FGFR3, FGFR4,FGFR5, Fgr, fit-1, Fps, Frk, Fyn, Hck, IGF-1R, INS-R, Jak, KDR, Lck,Lyn, MEK, p38, PDGFR, PIK, PKC, PYK2, ros, Tie, Tie-2, TRK, Yes, andZap70.

Studies indicated that protein kinases play a central role in theregulation and maintenance of a wide variety of cellular processes andcellular function. For example, kinase activity acts as molecularswitches regulating cell proliferation, activation, and/ordifferentiation. Uncontrolled or excessive kinase activity has beenobserved in many disease states including benign and malignantproliferation disorders as well as diseases resulting from inappropriateactivation of the immune system (autoimmune disorders), allograftrejection, and graft vs host disease.

It is reported that many diseases are associated with abnormal cellularresponses triggered by protein kinase-mediated events. These diseasesinclude autoimmune diseases, inflammatory diseases, bone diseases,metabolic diseases, neurological and neurodegenerative diseases, cancer,cardiovascular diseases, allergies and asthma, Alzheimer's disease andhormone-related diseases. In addition, endothelial cell specificreceptor PTKs, such as VEGF-2 and Tie-2, mediate the angiogenic processand are involved in supporting the progression of cancers and otherdiseases involving uncontrolled vascularization. Accordingly, there hasbeen a substantial effort in medicinal chemistry to find protein kinaseinhibitors that are effective as therapeutic agents.

Many cancers are characterized by distruptions in cellular signalingpathways that lead to uncontrolled growth and proliferation of cancerouscells. Receptor tyrosine kinases (RTKs) play a crucial role in thesesignaling pathways, transmitting extracellular molecular signals intocytoplasm and/or nucleus of a cell. RTKs are transmembrane proteins thatgenerally include an extracellular ligand-binding domain, amembrane-spanning domain and a catalytic cytoplasmic tyrosine kinasedomain. The binding of ligand to the extracellular potion is believed topromote dimerization, resulting in trans-phosphorylation and activationof the intracellular tyrosine kinase domain (Schlessinger et al. Neuron1992; 9:383-391).

Considering the lack of currently available treatment options for themajority of the conditions associated with protein kinases, there isstill a great need for new therapeutic agents that inhibit these proteintargets.

SUMMARY OF THE INVENTION

Accordingly, it is an objective of the present invention to provide anantitumor agent comprising a substituted indol-5-ol derivatives asdescribed in formula (I), pharmaceutically-acceptable formulationsthereof, methods for making novel compounds and compositions for usingthe compounds. The compounds and compositions comprising the compoundsin formula (I) have utility in treatment of a variety of diseases.

The combination therapy described herein may be provided by thepreparation of the substituted indol-5-ol derivatives of formula (I) andthe other therapeutic agent as separate pharmaceutical formulationsfollowed by the administration thereof to a patient simultaneously,semi-simultaneously, separately or over regular intervals.

The present invention provides methods of use for certain chemicalcompounds such as kinase inhibitors for treatment of various diseases,disorders, and pathologies, for example, cancer, and vascular disorders,such as myocardial infarction (MI), stroke, or ischemia. The triazinecompounds described in this invention may block the enzymatic activityof some or many of the members of the Aurora kinase family, in additionto blocking the activity of other receptor and non-receptor kinase. Suchcompounds may be beneficial for treatment of the diseases wheredisorders affect cell motility, adhesion, and cell cycle progression,and in addition, diseases with related hypoxic conditions, osteoporosisand conditions, which result from or are related to increases invascular permeability, inflammation or respiratory distress, tumorgrowth, invasion, angiogenesis, metastases and apoptosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the kinase inhibitory activity of NTW-3475 against a widerange of kinases from a wide range of diseases.

FIG. 2 depicts the kinase inhibitory activity of NTW-3475 against a widerange of mutant kinases.

FIG. 3 depicts the in vitro antiproliferation activity of NTW-3475.

FIG. 4 depicts the animal's body weight change in a xenograft study thetreatment of Acute Myleogenous Leukemia (AML) with NTW-3475 (body weightchange is a marker for overall toxicity.)

FIG. 5 depicts the tumor size curve for NTW-3475 in the AML study,showing anti-tumor activity.

FIG. 6 depicts the relative antitumor activity of NTW-3475 in terms ofsize of tumor in animals treated with NTW-3475/size of the AML tumorgiven only expose to negative control agents.

FIG. 7 depicts the animals' body weight change in the xenograft study ofthe utility of NTW-3475 in treating Chronic Myelogenous Leukemia (CML).

FIG. 8 depicts the tumor curve v. time for NTW-3475 treated and controlmice in a xenograft study of Pancreatic Carcinoma.

FIG. 9 depicts animals' body weight change in the xenograft withNTW-3475 (a marker for overall toxicity).

FIG. 10 depicts the relative antiproliferative activity of NTW-3475 interms of size of tumor in animals treated/size of tumor with NTW-3475 ora control agent.

FIG. 11. depicts the relative antiproliferative activity of NTW-3475 interms of size of tumors in animals treated with NTW-3475/size of tumorwith a control agent.

FIG. 12 depicts the weight curve for NTW-3475, in a study of its use inthyroid carcinoma.

FIG. 13 depicts the tumor size v. time curve for NTW-3475 treated,inoculate in the morning showing anti-tumor activity. In the thyroidstudy.

FIG. 14. depicts xenograft containing animals' weight while undergoingtreatment with NTW-3475 alone or in combination with nanoparticulatealbumin bound paclitaxel (Abraxane®) in a study of Endometrial carcinomausing a xenograft model.

FIG. 15. depicts the tumor size curves for NTW-3475 with or withoutAbraxane® in that study of Endometrial carcinoma.

FIG. 16. depicts the relative antiproliferative activity of NTW-3475 andAbraxane®) in terms of size of tumor in animals treated/size of tumor inmice given a control agent from that study of Endometrial carcinomamodel.

FIG. 17. depicts effect on animals' weight (a marker for overalltoxicity) while undergoing Abraxane®-NTW-3475 combination therapy ofPancreatic Carcinoma using a xenograft model.

FIG. 18. depicts the tumor size curve for NTW-3475 and thenanoparticulate, albumin bound paclitaxel, Abraxane®, PancreaticCarcinoma using a xenograft model.

FIG. 19 depicts the % T/C for NTW-3475 and Abraxane®, in combinationtherapy using a xenograft model of pancreatic carcinoma.

FIG. 20 summarizes NTW-3475's in vitro activity.

FIG. 21 Summarizes the in vivo (xenografts) pharmacology of NTW-3475.

FIG. 22. depicts the kinase inhibitory activity of NTW-3456 against awide range of kinases in a wide range of cancers.

FIG. 23. depicts the kinase inhibitory activity of NTW-3475 against awide range of mutant kinases.

FIG. 24 depicts the kinase inhibitory activity of NTW-3456 againstmutant abl kinases.

FIG. 25. depicts the in vitro antiproliferation activity of NTW-3456.

FIG. 26. Depicts exemplary antiproliferative and phosphorylationinhibition activity for NTW-34-56.

FIG. 27. depicts the weight time course for AML xenograft containinganimals' weight while undergoing NTW-3456 (a marker for overalltoxicity.)

FIG. 28. depicts the tumor size curve for NTW-3456 treated animals,showing anti-tumor activity in an AML model system.

FIG. 29. depicts the antitumor activity of NTW-3456 in terms of size oftumor size in treated/size of tumor in untreated.

FIG. 30. depicts the weight time course for AML xenograft containinganimals' weight while undergoing NTW-3456 (a marker for overalltoxicity.)

FIG. 31. depicts the tumor size curve for NTW-3456 treated animals,showing anti-tumor activity in an AML model system.

FIG. 32. depicts the tumor activity of NTW-3456 in terms of size oftumor size in treated/size of tumor in untreated. (FIGS. 27-29 and 30-32are separate experiments).

FIG. 33. Demonsrates the correlation between PK and inhibition of pFlt3in SCID mice bearing MV4-11 human acute myeloid leukemia following asingle oral administration of 50 mg/kg NTW-3456.

FIG. 34. depicts the tumor size curve for NTW-3456 treated animals,showing anti-tumor activity in a CML model system.

FIG. 35. depicts the tumor activity of NTW-3456 in terms of size oftumor size in treated/size of tumor in untreated in a CML model system.

FIG. 36. depicts the weight time course for animals' weight whileundergoing NTW-3456 (a marker for overall toxicity) in a thyroidcarcinoma model system.

FIG. 37. depicts the tumor size curve for NTW-3456 treated animals,showing anti-tumor activity in a thyroid carcinoma model system.

FIG. 38. depicts the tumor activity of NTW-3456 in terms of size oftumor size in treated/size of tumor in untreated animals in a thyroidcarcinoma model system.

FIG. 39. depicts the weight time course for animals' weight whileundergoing NTW-3456 (a marker for overall toxicity) in a endometrialcarcinoma model system.

FIG. 40. depicts the tumor size curve for NTW-3456 treated animals,showing anti-tumor activity in a endometrial carcinoma model system.

FIG. 41. depicts the tumor activity of NTW-3456 in terms of size oftumor size in treated/size of tumor in untreated animals in aendometrial carcinoma model system.

FIG. 42. depicts the weight time course for animals' weight whileundergoing NTW-3456 (a marker for overall toxicity) in a pancreaticcarcinoma model system.

FIG. 43. depicts the tumor size curve for NTW-3456 treated animals,showing anti-tumor activity in a pancreatic carcinoma model system.

FIG. 44. depicts the tumor activity of NTW-3456 in terms of size oftumor size in treated/size of tumor in untreated animals in a pancreaticcarcinoma model system.

FIG. 45. depicts the weight time course for containing animals' weightwhile undergoing NTW-3456 single or dual agent therapy with Abraxane® (amarker for overall toxicity) in a MiaPaCa-2 pancreatic carcinoma modelsystem.

FIG. 46. depicts the tumor size curve for NTW-3456 single or dual agenttherapy with Abraxane treated animals, showing anti-tumor activity in aMiaPaCa-2 pancreatic carcinoma model system.

FIG. 47. depicts the antitumor activity of NTW-3456 alone or withAbraxane® in terms of size of tumor size in treated/size of tumor inuntreated animals in a in a MiaPaCa-2 pancreatic carcinoma model system.

FIG. 48. depicts the weight time course for containing animals' weightwhile undergoing NTW-3456 single or dual agent therapy with Abraxane® (amarker for overall toxicity) in a Panc-1 pancreatic carcinoma modelsystem.

FIG. 49. depicts the tumor size curve for NTW-3456 single or dual agenttherapy with Abraxane® treated animals, showing anti-tumor activity in aPanc-1 pancreatic carcinoma model system.

FIG. 50 summarizes the in vivo pharmacology of NTW-3456.

FIG. 51 shows the signal transduction effects of NTW-3456 on MiaPaCa-2cells and BxPC3 cells.

FIG. 52 shows the signal transduction effects of NTW-3475 in MiaPaCa-2cells and BxPC3 cells.

FIG. 53 summarizes the inhibitory activities of NTW-3456 and NTW-3475.

FIG. 54 depicits the inhibitory activities of NTW-3456 on the growth ofK562 cells.

FIG. 55 depicits the inhibitory activities of NTW-3456 on pCrlinhibition in K562 cells.

FIG. 56 depicits the inhibitory activities of NTW-3456 on caspase 3/7induction in K562 cells.

FIG. 57 summarizes the inhibitory activities of NTW-3456.

FIG. 58 shows the NTW-3456 dose response curve for the inhibition of theFGFR1-FGFR-4 in BaF3 cells.

FIG. 59 summarizes the inhibitory activities of NTW-3456 on FibroblastGrowth Factor Receptors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to compounds having general Formula (I)

or a pharmaceutically acceptable salt thereof, wherein:

Z1, Z2, Z3, and Z4 is independently N or as described below;

R is selected from:

(i) hydrogen, amino, alkyl amino;

(ii) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl;

(iii) K—Ar.

Ar represents heteroaryl or aryl, each of which is substituted with from0 to 4 substituents independently chosen from:

(1) halogen, hydroxy, amino, amide, cyano, —COOH, —SO2NH2, oxo, nitroand alkoxycarbonyl; and

(2) C1-C6 alkyl, C1-C6 alkoxy, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6alkynyl, C2-C6 alkanoyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, mono- anddi-(C1-C6alkyl)amino, C1-C6 alkylsulfonyl, mono- and di-(C1-C6alkyl)sulfonamido and mono- and di-(C1-C6alkyl)aminocarbonyl; phenylC0-C4alkyland (4- to 7-membered heterocycle)C0-C4alkyl, each of which issubstituted with from 0 to 4 secondary substituents independently chosenfrom halogen, hydroxy, cyano, oxo, imino, C1-C4 alkyl, C1-C4 alkoxy andC1-C4 haloalkyl.

K is selected from

-   -   a) O, S, SO, SO2;    -   b) (CH2)m, m=0-3, —O(CH2)p, p=1-3, —S(CH2)p, p=1-3, —N(CH2)p,        p=1-3, —(CH2)pO, p=1-3;    -   c) NR1    -   R1 represents hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,        alkylthio, aryl, arylalkyl.    -   (iv) groups of the formula (Ia):

wherein:

R₂ represents hydrogen, C₁-C₄ alkyl, oxo;

X is CH, when R₃ is hydrogen; or X—R₃ is O; or X is N, R₃ representsgroups of hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀aryl or heteroaryl, (C₃-C₇cycloalkyl)C₁-C₄ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkoxy, C₁-C₆ alkylthio, C₂-C₆ alkanoyl, C₁-C₆ alkoxycarbonyl, C₂-C₆alkanoyloxy, mono- and di-(C₃-C₈ cycloalkyl)aminoC₀-C₄alkyl, (4 to7-membered heterocycle)C₀-C₄alkyl, C₁-C₆ alkylsulfonyl, mono- anddi-(C₁-C₆ alkyl) sulfonamido, and mono- and di-(C₁-C₆alkyl)aminocarbonyl, each of which is substituted with from 0 to 4substituents independently chosen from halogen, hydroxy, cyano, amino,—COOH and oxo.

R₁₁ and R₁₂ are independently selected from: Hydrogen, F, Cl, Br, CN,C₁-C₄ alkyl, C₁-C₆ alkoxy.

R₁₃, R₁₄ and R₁₅ are independently selected from Hydrogene, C₁-C₄ alkyl,C₂-C₆ alkenyl, CF₃, CF₂H, CFH₂, C₂-C₆ alkynyl, C₃-C₁₀ aryl orheteroaryl, C₁-C₆ alkoxy, C₁-C₆ alkylthio, C₂-C₆ alkanoyl, C₁-C₆alkoxycarbonyl, C₂-C₆ alkanoyloxy.

Ring A is selected from the group consisting of:

R_(x) and R_(y)— are independently selected from T-R₄, or R_(x) and—R_(y) are taken together with their intervening atoms to form a fused,unsaturated or partially unsaturated, 5-7 membered ring having 0-3 ringheteroatoms selected from oxygen, sulfur, or nitrogen, wherein anysubstitutable carbon on said fused ring formed by R_(x) and R_(y) issubstituted by oxo or T-R₄, and any substitutable nitrogen on said ringformed by R_(x) and R_(y) is substituted by R₅;

T is a valence bond or a C₁₋₄ alkylidene chain;

R4 is selected from —R6, -halo, —OR6, —C(═O)R6, —CO2R6, —COCOR6,—COCH2COR6, —NO2, —CN, —S(O)R6, —S(O)2R6, —SR6, —N(R5)2, —CON(R7)2,SO2N(R7)2, —OC(═O)R6, —N(R7)COR6, —N(R7)CO2 (optionally substituted C1-6aliphatic), —N(R5)N(R5)2, —C═NN(R5)2, —C═N—OR6, —N(R7)CON(R7)2, —N(R7)SO2N(R7)2, —N(R4) SO2R6, or —OC(═O)N(R7)2;

Each R₆ is independently selected from hydrogen or an optionallysubstituted group selected from C₁₋₆ aliphatic, C₆₋₁₀ aryl, a heteroarylring having 5-10 ring atoms, or a heterocyclyl ring having 5-10 ringatoms;

Each R₅ is independently selected from —R₇, —COR₇, —CO₂(C₁₋₆ aliphatic),CON(R₇)₂, or —SO₂R₇, or two R₅ on the same nitrogen are taken togetherto form a 5-8 membered heterocyclyl or heteroaryl ring;

Each R₇ is independently selected from hydrogen or an optionallysubstituted C₁₋₆ aliphatic group, or two R₇ on the same nitrogen aretaken together with the nitrogen to form a 5-8 membered heterocyclyl orheteroaryl ring;

R₈ is selected from —R₆, halo, —OR₆, —C(═O)R₆, —CO₂R₆, —COCOR₆, —NO₂,—CN, —S(O)R₆, —SO₂R₆, —SR₆, —N(R₄)₂, —CON(R₅)₂, —SO₂N(R₅)₂, —OC(═O)R₆,—N(R₅)COR₆, —N—(R₅)CO₂ (optionally substituted C₁₋₆ aliphatic),—N(R₅)N(R₅)₂, —C═NN(R₅)₂, —C═N—OR₆, —N(R₅)CON(R₅)₂, —N(R₅)SO₂N(R₅)₂,—N(R₅)SO₂R₆, or —OC(═O)N(R₅)₂.

R_(x) and R_(y) (at positions Z₃ and Z₄, respectively) may be takentogether to form a fused ring, providing a bicyclic ring systemcontaining Ring A. alternatively, R and Z₂ also may be taken together toform a fused ring, providing a bicyclic ring system containing Ring A.Preferred R_(x)/R_(y) and R/Z₂ rings include a 5-, 6-, or 7-, memberedunsaturated or partially unsaturated ring having 0-2 heteroatoms,wherein said R_(x)/R_(y) and R/Z₂ ring is optionally substituted.Examples of Ring A systems are shown below by compounds I-1 throughI-26, wherein Z₁ to Z₄ is nitrogen or C(R₈).

Preferred bicyclic Ring A systems include I-1, I-2, I-3, I-4, I-5, I-6,I-7, I-8, I-14, I-15 I-16, I-17, I-19, I-23 and I-24, more preferablyI-1, I-2, I-3, I-5, I-8, I-14, I-15 I-16, I-17, I-19, I-23 and I-24, andmost preferably I-1, I-14, I-16, and I-19.

In the monocyclic Ring A system, preferred R^(x) groups, when present,include hydrogen, alkyl- or dialkylamino, acetamido, or a C₁₋₄ aliphaticgroup such as methyl, ethyl, cyclopropyl, isopropyl or t-butyl.Preferred R^(y) groups, when present, include T-R₄ wherein T is avalence bond or a methylene, and R₄ is —R₆, —N(R₅)₂, or —OR₆. Examplesof preferred R^(y) include 2-pyridyl, 4-pyridyl, piperidinyl, methyl,ethyl, cyclopropyl, isopropyl, t-butyl, alkyl- or dialkylamino,acetamido, optionally substituted phenyl such as phenyl orhalo-substituted phenyl, and methoxymethyl.

In the bicyclic Ring A system, the ring formed when R^(x) and R^(y) aretaken together may be substituted or unsubstituted. Suitablesubstituents include —R₆, halo, —OR₆, —C(═O)R₆, —CO₂R₆, —COCOR₆, —NO₂,—CN, —S(O)R₆, —SO₂R₆, —SR₆, —N(R₅)₂, —CON(R₅)₂, —SO₂N(R₅)₂, —OC(═O)R₆,—N(R₄)COR₆, —N(R₅)CO₂ (optionally substituted C₁₋₆ aliphatic),—N(R₅)N(R₅)₂, —C═NN(R₅)₂, —C═N—OR₆, —N(R₅)CON(R₅)₂, —N(R₅)SO₂N(R₅)₂,—N(R₅)SO₂R₆ or —OC(═O)N(R₅)₂, wherein R and R⁵ are as defined above.Preferred R^(x)/R^(y) ring substituents include -halo, —R₆, —OR₆, —COR₆,—CO₂R₆, —CON(R₅)₂, —CN, or —N(R₅)₂ wherein R6 is hydrogen or anoptionally substituted C₁₋₆ aliphatic group.

An embodiment that is particularly useful for treating kinase-mediateddiseases relates to compounds of formula IIa, IIb and IIc:

-   -   or a pharmaceutically acceptable derivative or prodrug thereof,        wherein;        R is selected from:

(i) hydrogen, amino, alkyl amino;

(ii) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl;

(iii) K—Ar.

Ar represents heteroaryl or aryl, each of which is substituted with from0 to 4 substituents independently chosen from:

(1) halogen, hydroxy, amino, amide, cyano, —COOH, —SO2NH2, oxo, nitroand alkoxycarbonyl; and

(2) C1-C6 alkyl, C1-C6 alkoxy, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6alkynyl, C2-C6 alkanoyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, mono- anddi-(C1-C6alkyl)amino, C1-C6 alkylsulfonyl, mono- and di-(C1-C6alkyl)sulfonamido and mono- and di-(C1-C6alkyl)aminocarbonyl; phenylC0-C4alkyland (4- to 7-membered heterocycle)C0-C4alkyl, each of which issubstituted with from 0 to 4 secondary substituents independently chosenfrom halogen, hydroxy, cyano, oxo, imino, C1-C4 alkyl, C1-C4 alkoxy andC1-C4 haloalkyl.

K is selected from

a) O, S, SO, SO2;

b) (CH2)m, m=0-3, —O(CH2)p, p=1-3, —S(CH2)p, p=1-3, —N(CH2)p, p=1-3,—(CH2)pO, p=1-3;

c) NR1

R₁ represents hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkylthio,aryl, arylalkyl.

(iv) groups of the formula (Ia):

-   -   wherein:    -   R₂ represents hydrogen, C₁-C₄ alkyl, oxo;    -   X is CH, when R₃ is hydrogen; or X—R₃ is O; or X is N, R₃        represents groups of hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₁₀ aryl or heteroaryl, (C₃-C₇cycloalkyl)C₁-C₄alkyl,        C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ alkylthio, C₂-C₆ alkanoyl,        C₁-C₆ alkoxycarbonyl, C₂-C₆ alkanoyloxy, mono- and di-(C₃-C₈        cycloalkyl)aminoC₀-C₄alkyl, (4 to 7-membered        heterocycle)C₀-C₄alkyl, C₁-C₆ alkylsulfonyl, mono- and di-(C₁-C₆        alkyl) sulfonamido, and mono- and di-(C₁-C₆alkyl)aminocarbonyl,        each of which is substituted with from 0 to 4 substituents        independently chosen from halogen, hydroxy, cyano, amino, —COOH        and oxo.

Het is selected from any heterocycle, which is substituted with from 0to 4 substituents independently chosen from:

(i) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl;

(ii) halogen, hydroxy, amino, amide, cyano, —COOH, —SO2NH2, oxo, nitroand alkoxycarbonyl,

(iii) aryl.

Substituents on indole are as the following:

R11 and R12 are independently selected from: Hydrogen, F, Cl, Br, CN,C1-C4 alkyl, C1-C6 alkoxy.

R₁₃, R₁₄ and R₁₅ are independently selected from Hydrogene, C₁-C₄ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀ aryl or heteroaryl, C₁-C₆ alkoxy,C₁-C₆ alkylthio, C₂-C₆ alkanoyl, C₁-C₆ alkoxycarbonyl, C₂-C₆alkanoyloxy.

Preferred R groups of formula (I) are listed below:

Preferred Het groups formula (II) are list below, wherein the substitutemay be the specific ones as defined here or may be one or multiplesubstitutes as defined above:

R′ is selected from

(i) Hydrogen;

(ii) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl;

(iii) aryl, which may have 1-4 optically substituents;

(iii) —C(═O)R6, (R6 is described below)

Preferred substituted indole groups of formula (I) are listed below:

Embodiments of the invention include:

In another embodiment, a method of preparing the inventive compounds isprovided. The compounds of the present invention can be generallyprepared using 4, 6-dichloro-2-methylsulfonylpyrimidine, or2,4,6-trichloropyrimidine, or 4,6-dichloro-2-(methylthio)pyrimidine as astarting material. Compound (I) may contain various stereoisomers,geometric isomers, tautomeric isomers, and the like. All of possibleisomers and their mixtures are included in the present invention, andthe mixing ratio is not particularly limited.

The pyrimidine derivative compounds of Formula (IIa, IIb and IIc) inthis invention can be synthesized from commercially available precursorsusing established protocols. By way of example, a synthetic routesimilar to that shown in any of the following Schemes may be used,together with synthetic methods known in the art of synthetic organicchemistry, or variations thereon as appreciated by those skilled in theart. Each variable in the following schemes refers to any groupconsistent with the description of the compounds provided herein.

In the Schemes that follow the term “reduction” refers to the process ofreducing a nitro functionality to an amino functionality, or the processof transforming an ester functionality to an alcohol. The reduction of anitro group can be carried out in a number of ways well known to thoseskilled in the art of organic synthesis including, but not limited to,catalytic hydrogenation, reduction with SnCI2 and reduction withtitanium bichloride. In the Schemes that follow, the term “hydrolyze”refers to the reaction of a substrate or reactant with water. Morespecifically, “hydrolyze” refers to the conversion of an ester ornitrite functionality into a carboxylic acid. This process can becatalyzed by a variety of acids or bases well known to those skilled inthe art of organic synthesis.

The compounds of Formula (IIa, IIb, and IIc) may be prepared by use ofknown chemical reactions and procedures. The following generalpreparative methods are presented to aid one of skill in the art insynthesizing the inhibitors, with more detailed examples being presentedin the experimental section describing the working examples.

Propenyl-pyrazol amine as defined in formula (III) is not commerciallyavailable. It can be prepared by several methods as described earlier(see, e.g., U.S. provisional application No. 61/555,738).

Precursors of substituted indol-5-ol as defined in formula (IV) can bepurchased from suppliers, or synthesized from commercially availableprecursors using established protocols. (WO 2004/009542, P33-38; Journalof Medicinal Chemistry, 2006, Vol 49, No. 7, P2143-2146; Org. Lett. Vol10, No 12, 2008, P 2369-2372; WO 00/47212, P245-250; WO 2009036055 A1,P57).

Especially, precursor 4-7-d-fluoroindol-5-ol as defined in formula (IVa)was not reported before and can be prepared with the same token.

For example, as illustrated in scheme 1, precursor (IV) can be preparedfrom the commercially available starting materials via several steps.Also various synthetic routs can be utilized to prepare the compound.

The preparation of the compounds of formula (IIa, IIb and IIc) in thisinvention can be carried out by methods known in the art.

As shown in scheme 2, the pyrimidine derivative (IIa) can be synthesizedby the reaction of 2,4,6-trichloropyrimidine, or4,6-dichloro-2-(methylsulfonyl)pyrimidine, with a sequence ofsustitutedidole-5-ol s to give dichiororopyrimidine intermediate ofcompound b, which can react with WH to produce the advancedmonochlorointenmediate of compound c. The displacement of the lastchlorine by RH can be achieved by increasing the temperature, affordingthe final compound (IIa). The reaction can be strpwise or in one pot.Alternative sequence can also be used to make pyrimidine derivatives.With the same token, Compound JIb and IIc can also be synthesized.

Scheme 2

The reaction is preferably conducted in the presence of an inertsolvent. There is no particular restriction on the nature of the solventto be employed, provided that it has no adverse effect on the reactionor on the reagents involved and that it can dissolve the reagents, atleast to some extent. Examples of suitable solvents include: aliphatichydrocarbons, such as hexane, heptane, ligroin and petroleum ether;aromatic hydrocarbons, such as benzene, toluene and xylene; halogenatedhydrocarbons, especially aromatic and aliphatic hydrocarbons, such asmethylene chloride, chloroform, carbon tetrachloride, dichloroethane,chlorobenzene and the dichlorobenzenes; esters, such as ethyl formate,ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate;ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran,dioxane. dimethoxyethane and diethylene glycol dimethyl ether; ketones,such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophoroneand cyclohexanone; nitro compounds, which may be nitroalkanes ornitroaranes, such as nitroethane and nitrobenzene; nitriles, such asacetonitrile and isobutyronitrile; amides, which may be fatty acidamides, such as formamide, dimethylformamide, dimethylacetamide andhexamethylphosphoric triamide; and sulphoxides, such as dimethylsulphoxide and sulpholane.

The reaction can take place over a wide range of temperatures, and theprecise reaction temperature is not critical to the invention. Ingeneral, we find it convenient to carry out the reaction at atemperature of from −50° C. to 100° C.

The present invention provides compositions of matter that areformulations of one or more active drugs and apharmaceutically-acceptable carrier. In this regard, the inventionprovides a composition for administration to a mammalian subject, whichmay include a compound of formula I, or its pharmaceutically acceptablesalts.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN+(C1-4 alkyl)4 salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, troches, elixirs, suspensions,syrups, wafers, chewing gums, aqueous suspensions or solutions.

The oral compositions may contain additional ingredients such as: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, corn starch and the like; a lubricant such as magnesiumstearate; a glidant such as colloidal silicon dioxide; and a sweeteningagent such as sucrose or saccharin or flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. When the dosage unitform is a capsule, it may additionally contain a liquid carrier such asa fatty oil. Other dosage unit forms may contain other various materialswhich modify the physical form of the dosage unit, such as, for example,a coating. Thus, tablets or pills may be coated with sugar, shellac, orother enteric coating agents. A syrup may contain, in addition to theactive ingredients, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically orveterinarally pure and non-toxic in the amounts used.

For the purposes of parenteral therapeutic administration, the activeingredient may be incorporated into a solution or suspension. Thesolutions or suspensions may also include the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

The pharmaceutical forms suitable for injectable use include sterilesolutions, dispersions, emulsions, and sterile powders. The final formshould be stable under conditions of manufacture and storage.Furthermore, the final pharmaceutical form should be protected againstcontamination and should, therefore, be able to inhibit the growth ofmicroorganisms such as bacteria or fungi. A single intravenous orintraperitoneal dose can be administered. Alternatively, a slowlong-term infusion or multiple short-term daily infusions may beutilized, typically lasting from 1 to 8 days. Alternate day dosing ordosing once every several days may also be utilized.

Sterile, injectable solutions may be prepared by incorporating acompound in the required amount into one or more appropriate solvents towhich other ingredients, listed above or known to those skilled in theart, may be added as required. Sterile injectable solutions may beprepared by incorporating the compound in the required amount in theappropriate solvent with various other ingredients as required.Sterilizing procedures, such as filtration, may then follow. Typically,dispersions are made by incorporating the compound into a sterilevehicle which also contains the dispersion medium and the required otheringredients as indicated above. In the case of a sterile powder, thepreferred methods include vacuum drying or freeze drying to which anyrequired ingredients are added.

Suitable pharmaceutical carriers include sterile water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil; liquid acid; lower alkanols; oils such ascorn oil; peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like; glycols; polyalkylene glycols; aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and thelike, alone, or with suitable dispensing agents such as lecithin;polyoxyethylene stearate; and the like. The carrier may also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer. In all cases, thefinal form, as noted, must be sterile and should also be able to passreadily through an injection device such as a hollow needle. The properviscosity may be achieved and maintained by the proper choice ofsolvents or excipients. Moreover, the use of molecular or particulatecoatings such as lecithin, the proper selection of particle size indispersions, or the use of materials with surfactant properties may beutilized.

In accordance with the invention, there are provided compositionscontaining triazine derivatives and methods useful for the in vivodelivery of triazine derivatives in the form of nanoparticles, which aresuitable for any of the aforesaid routes of administration.

U.S. Pat. Nos. 5,916,596, 6,506,405 and 6,537,579 teach the preparationof nanoparticles from the biocompatible polymers, such as albumin. Thus,in accordance with the present invention, there are provided methods forthe formation of nanoparticles of the present invention by a solventevaporation technique from an oil-in-water emulsion prepared underconditions of high shear forces (e.g., sonication, high pressurehomogenization, or the like).

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

In accordance with the invention, the compounds of the invention may beused to treat diseases associated with cellular proliferation orhyperproliferation, such as cancers which include but are not limited totumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity,oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas.The compounds of the invention may also be used to treat cancers of theliver and biliary tree (particularly hepatocellular carcinoma),intestinal cancers, particularly colorectal cancer, ovarian cancer,small cell and non-small cell lung cancer, breast cancer, sarcomas(including fibrosarcoma, malignant fibrous histiocytoma, embryonalrhabdomysocarcoma, leiomysosarcoma, neuro-fibrosarcoma, osteosarcoma,synovial sarcoma, liposarcoma, and alveolar soft part sarcoma),neoplasms of the central nervous systems (particularly brain cancer),and lymphomas (including Hodgkin's lymphoma, lymphoplasmacytoidlymphoma, follicular lymphoma, mucosa-associated lymphoid tissuelymphoma, mantle cell lymphoma, B-lineage large cell lymphoma, Burkitt'slymphoma, and T-cell anaplastic large cell lymphoma).

The compounds and methods of the present invention, either whenadministered alone or in combination with other agents (e.g.,chemotherapeutic agents or protein therapeutic agents described below)are also useful in treating a variety of disorders, including but notlimited to, for example: stroke, cardiovascular disease, myocardialinfarction, congestive heart failure, cardiomyopathy, myocarditis,ischemic heart disease, coronary artery disease, cardiogenic shock,vascular shock, pulmonary hypertension, pulmonary edema (includingcardiogenic pulmonary edema), pleural effusions, rheumatoid arthritis,diabetic retinopathy, retinitis pigmentosa, and retinopathies, includingdiabetic retinopathy and retinopathy of prematurity, inflammatorydiseases, restenosis, asthma, acute or adult respiratory distresssyndrome (ARDS), lupus, vascular leakage, protection from ischemic orreperfusion injury such as ischemic or reperfusion injury incurredduring organ transplantation, transplantation tolerance induction;ischemic or reperfusion injury following angioplasty; arthritis (such asrheumatoid arthritis, psoriatic arthritis or osteoarthritis); multiplesclerosis; inflammatory bowel disease, including ulcerative colitis andCrohn's disease; lupus (systemic lupus crythematosis); graft vs. hostdiseases; T-cell mediated hypersensitivity diseases, including contacthypersensitivity, delayed-type hypersensitivity, and gluten-sensitiveenteropathy (Celiac disease); Type 1 diabetes; psoriasis; contactdermatitis (including that due to poison ivy); Hashimoto's thyroiditis;Sjogren's syndrome; Autoimmune Hyperthyroidism, such as Graves' disease;Addison's disease (autoimmune disease of the adrenal glands); autoimmunepolyglandular disease (also known as autoimmune polyglandular syndrome);autoimmune alopecia; pernicious anemia; vitiligo; autoimmunehypopituatarism; Guillain-Barre syndrome; other autoimmune diseases;cancers, including those where kineses such as Src-family kineses areactivated or overexpressed, such as colon carcinoma and thymoma, orcancers where kinase activity facilitates tumor growth or survival;glomerulonephritis, serum sickness; uticaria; allergic diseases such asrespiratory allergies (asthma, hayfever, allergic rhinitis) or skinallergies; mycosis fungoides; acute inflammatory responses (such asacute or adult respiratory distress syndrome and ischemialreperfusioninjury); dermnatomyositis; alopecia areata; chronic actinic dermatitis;eczema; Behcet's disease; Pustulosis palmoplanteris; Pyoderma gangrenum;Sezary's syndrome; atopic dermatitis; systemic schlerosis; morphea;peripheral limb ischemia and ischemic limb disease; bone disease such asosteoporosis, osteomalacia, hyperparathyroidism, Paget's disease, andrenal osteodystrophy; vascular leak syndromes, including vascular leaksyndromes induced by chemotherapies or immunomodulators such as IL-2;spinal cord and brain injury or trauma; glaucoma; retinal diseases,including macular degeneration; vitreoretinal disease; pancreatitis;vasculatides, including vasculitis, Kawasaki disease, thromboangiitisobliterans, Wegener s granulomatosis, and Behcet's disease; scleroderma;preeclampsia; thalassemia; Kaposi's sarcoma; von Hippel Lindau disease;and the like.

In accordance with the invention, the compounds of the invention may beused to treat diseases associated with undesired cellular proliferationor hyperproliferation comprising identifying the mammal afflicted withsaid disease or condition and administering to said afflicted mammal acomposition comprising the compound of formula 1, wherein the disease orcondition is associated with a kinase.

In accordance with the invention, the compounds of the invention may beused to treat diseases associated with undesired cellular proliferationor hyperproliferation comprising identifying the mammal afflicted withsaid disease or condition and administering to said afflicted mammal acomposition comprising the compound of formula 1, wherein the disease orcondition is associated with a tyrosine kinase.

In accordance with the invention, the compounds of the invention may beused to treat diseases associated with undesired cellular proliferationor hyperproliferation comprising identifying the mammal afflicted withsaid disease or condition and administering to said afflicted mammal acomposition comprising the compound of formula 1, wherein the disease orcondition is associated with the kinase that is a serine kinase or athreonine kinase.

In accordance with the invention, the compounds of the invention may beused to treat diseases associated with undesired cellular proliferationor hyperproliferation comprising identifying the mammal afflicted withsaid disease or condition and administering to said afflicted mammal acomposition comprising the compound of formula 1, wherein the disease orcondition is associated with the kinase that is a Src family kinase.

The invention also provides methods of treating a mammal afflicted withthe above diseases and conditions. The amount of the compounds of thepresent invention that may be combined with the carrier materials toproduce a composition in a single dosage form will vary depending uponthe host treated, the particular mode of administration. Preferably, thecompositions should be formulated so that a dosage of between 0.01-100mg/kg body weight/day of the inhibitor can be administered to a patientreceiving these compositions.

In one aspect, the invention compounds are administered in combinationwith chemotherapeutic agent, an anti-inflammatory agent, antihistamines,chemotherapeutic agent, immunomodulator, therapeutic antibody or aprotein kinase inhibitor, e.g., a tyrosine kinase inhibitor, to asubject in need of such treatment.

The method includes administering one or more of the inventive compoundsto the afflicted mammal. The method may further include theadministration of a second active agent, such as a cytotoxic agent,including alkylating agents, tumor necrosis factors, intercalators,microtubulin inhibitors, and topoisomerase inhibitors. The second activeagent may be co-administered in the same composition or in a secondcomposition. Examples of suitable second active agents include, but arenot limited to, a cytotoxic drug such as Acivicin; Aclarubicin;Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine;Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine;Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa;Azotomycin; Batimastat; Benzodepa; Bicalutamide; BisantreneHydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate;Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflomithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride;Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate;Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine;Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; IdarubicinHydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; InterferonAlfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-□a;Interferon Gamma-Ib; Iproplatin; Irinotecan Hydrochloride; LanreotideAcetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride;Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol;Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine;Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide;Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper;Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan;Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium;Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin;Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safmgol;Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;Sparsomycin; Spirogennanium Hydrochloride; Spiromustine; Spiroplatin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; and ZorubicinHydrochloride.

In accordance with the invention, the compounds and compositions may beused at sub-cytotoxic levels in combination with other agents in orderto achieve highly selective activity in the treatment of non-neoplasticdisorders, such as heart disease, stroke and neurodegenerative diseases(Whitesell et al., Curr Cancer Drug Targets (2003), 3(5), 349-58).

The exemplary therapeutical agents that may be administered incombination with invention compounds include EGFR inhibitors, such asgefitinib, erlotinib, and cetuximab. Her2 inhibitors include canertinib,EKB-569, and GW-572016. Also included are Src inhibitors, dasatinib, aswell as Casodex (bicalutamide), Tamoxifen, MEK-1 kinase inhibitors, MARKkinase inhibitors, PI3 inhibitors, and PDGF inhibitors, such asimatinib, Hsp90 inhibitors, such as 17-AAG and 17-DMAG. Also includedare anti-angiogenic and antivascular agents which, by interrupting bloodflow to solid tumors, render cancer cells quiescent by depriving them ofnutrition. Castration, which also renders androgen dependent carcinomasnon-proliferative, may also be utilized. Also included are IGF1Rinhibitors, inhibitors of non-receptor and receptor tyrosine kineses,and inhibitors of integrin.

The pharmaceutical composition and method of the present invention mayfurther combine other protein therapeutic agents such as cytokines,immunomodulatory agents and antibodies. As used herein the term“cytokine” encompasses chemokines, interleukins, lymphokines, monokines,colony stimulating factors, and receptor associated proteins, andfunctional fragments thereof. As used herein, the term “functionalfragment” refers to a polypeptide or peptide which possesses biologicalfunction or activity that is identified through a defined functionalassay. The cytokines include endothelial monocyte activating polypeptideII (EMAP-II), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF(G-CSF), macrophage-CSF (M-CSF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-12, and IL-13, interferons, and the like and which is associated witha particular biologic, morphologic, or phenotypic alteration in a cellor cell mechanism.

Other therapeutic agents for the combinatory therapy includecyclosporins (e.g., cyclosporin A), CTLA4-Ig, antibodies such as ICAM-3,anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2, anti-CD3 (OKT-3),anti-CD4, anti-CD80, anti-CD86, agents blocking the interaction betweenCD40 and gp39, such as antibodies specific for CD40 and for gpn39 (i.e.,CD154), fusion proteins constructed from CD40 and gp39 (CD40Ig andCD8gp39), inhibitors, such as nuclear translocation inhibitors, ofNF-kappa B function, such as deoxyspergualin (DSG), cholesterolbiosynthesis inhibitors such as HM:G CoA reductase inhibitors(lovastatin and simvastatin), non-steroidal antiinflammatory drugs(NSAIDs) such as ibuprofen and cyclooxygenase inhibitors such asrofecoxib, steroids such as prednisone or dexamethasone, gold compounds,antiproliferative agents such as methotrexate, FK506 (tacrolimus,Prograf), mycophenolate mofetil, cytotoxic drugs such as azathioprineand cyclophosphamide, TNF-a inhibitors such as tenidap, anti-TNFantibodies or soluble TNF receptor, and rapamycin (sirolimus orRapamune) or derivatives thereof.

When other therapeutic agents are employed in combination with thecompounds of the present invention they may be used for example inamounts as noted in the Physician Desk Reference (PDR) or as otherwisedetermined by one having ordinary skill in the art.

The invention also encompasses pharmaceutical compositions comprisingany one or more of the compounds disclosed herein and said compounds inthe form of pharmaceutically acceptable salts, hydrates, solvates,crystal forms and individual steroisomers thereof (e.g, diastereomers,enantamers), and a in a compostion with a pharmaceutically acceptablecarrier. These include, but are not limited to, wherein the inventivecompounds are formulated into a composition in a neutral or salt form.

“Pharmaceutically acceptable salts” include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such as organic acids as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups also can bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, and procaine and the like.

“Salts” are chemical combinations of two ionizable components (e.g.,when dissolved in water), one acidic and the other basic with respect toone another. If in a salt form, a drug can be either the acidic or thebasic component.

“Pharmaceutically acceptable salts” include any salt form of thecompound wherein the salt is safe for animal ingestion (e.g., nontoxicto humans when taken orally). Exemplary such salts that can be used inaccordance with the invention include, but are not limited to,2-hydroxyethanesulfonate, 2-naphthalenesulfonate,3-hydroxy-2-naphthoate, 3-phenylpropionate, acetate, adipate, alginate,amsonate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate,bisulfate, bitartrate, borate, butyrate, calcium edetate, camphorate,camphorsulfonate, camsylate, carbonate, citrate, clavulariate,cyclopentanepropionate, digluconate, dodecylsulfate, edetate, edisylate,estolate, esylate, ethanesulfonate, finnarate, gluceptate,glucoheptanoate, gluconate, glutamate, glycerophosphate,glycollylarsanilate, hemisulfate, heptanoate, hexafluorophosphate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, laurylsulphonate, malate, maleate, mandelate,mesylate, methanesulfonate, methylbromide, methylnitrate, methylsulfate,mucate, naphthylate, napsylate, nicotinate, nitrate, N-methylglucamineammonium salt, oleate, oxalate, palmitate, pamoate, pantothenate,pectinate, persulfate, phosphate, phosphateldiphosphate, picrate,pivalate, polygalacturonate, propionate, p-toluenesulfonate, saccharate,salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate,suramate, tannate, tartrate, teoclate, thiocyanate, tosylate,triethiodide, undecanoate, and valerate salts, and the like (see also S.M. Berge et al., Pharmaceutical Salts, J. Pharm. Scis., 1977, 66:1-18;P. L. Gould, Salt selection for basic drugs, Int'l J. Pharms. 1986,33:201-17.)

“Solvates” are compositions which, during the process of crystallizationof a compound from solution, trap molecues of the solvent in the forminglattice.

“Hydrates” are solvates wherein the solvent was watter.

“Crystal” forms are solid compostions wherein the molecules making upthe compostion are packed in a repeating lattice structure. When morethan one lattice pattern is possible for compostions made up the samemolecules, the different compositions are called “polymorphs.”

“Diastereomers” are stereoisomers that are not related as object andmirror image, but still differ is in the arrangement inthree-dimensional space about one tetrahedral, sp3-hybridized carbon.

An “enantiomer” is one of two stereoisomers that are mirror images ofeach other, but are non-superposable (not identical).

“Pharmaceutically acceptable carrier” is any excipient which isnon-toxic and aids in a drug's function (see also, Rowe R C et al.,Handbook of Pharmaceutical Excipients, 5^(th) ed., 2006.)

EXAMPLES

The invention also encompasses pharmaceutical compositions comprisingany one or more of the compounds disclosed herein and said compounds inthe form of pharmaceutically acceptable salts, hydrates, solvates,crystal forms and individual steroisomers thereof (e.g, diastereomers,enantamers), and a in a compostion with a pharmaceutically acceptablecarrier. These include, but are not limited to, wherein the inventivecompounds are formulated into a composition in a neutral or salt form.

“Pharmaceutically acceptable salts” include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such as organic acids as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups also can bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, and procaine and the like.

“Salts” are chemical combinations of two ionizable components (e.g.,when dissolved in water), one acidic and the other basic with respect toone another. If in a salt form, a drug can be either the acidic or thebasic component.

“Pharmaceutically acceptable salts” include any salt form of thecompound wherein the salt is safe for animal ingestion (e.g., nontoxicto humans when taken orally). Exemplary such salts that can be used inaccordance with the invention include, but are not limited to,2-hydroxyethanesulfonate, 2-naphthalenesulfonate,3-hydroxy-2-naphthoate, 3-phenylpropionate, acetate, adipate, alginate,amsonate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate,bisulfate, bitartrate, borate, butyrate, calcium edetate, camphorate,camphorsulfonate, camsylate, carbonate, citrate, clavulariate,cyclopentanepropionate, digluconate, dodecylsulfate, edetate, edisylate,estolate, esylate, ethanesulfonate, finnarate, gluceptate,glucoheptanoate, gluconate, glutamate, glycerophosphate,glycollylarsanilate, hemisulfate, heptanoate, hexafluorophosphate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, laurylsulphonate, malate, maleate, mandelate,mesylate, methanesulfonate, methylbromide, methylnitrate, methylsulfate,mucate, naphthylate, napsylate, nicotinate, nitrate, N-methylglucamineammonium salt, oleate, oxalate, palmitate, pamoate, pantothenate,pectinate, persulfate, phosphate, phosphateldiphosphate, picrate,pivalate, polygalacturonate, propionate, p-toluenesulfonate, saccharate,salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate,suramate, tannate, tartrate, teoclate, thiocyanate, tosylate,triethiodide, undecanoate, and valerate salts, and the like (see also S.M. Berge et al., Pharmaceutical Salts, J. Pharm. Scis., 1977, 66:1-18;P. L. Gould, Salt selection for basic drugs, Int'l J. Pharms. 1986,33:201-17.)

“Solvates” are compositions which, during the process of crystallizationof a compound from solution, trap molecues of the solvent in the forminglattice.

“Hydrates” are solvates wherein the solvent was watter.

“Crystal” forms are solid compostions wherein the molecules making upthe compostion are packed in a repeating lattice structure. When morethan one lattice pattern is possible for compostions made up the samemolecules, the different compositions are called “polymorphs.”

“Diastereomers” are stereoisomers that are not related as object andmirror image, but still differ is in the arrangement inthree-dimensional space about one tetrahedral, sp3-hybridized carbon.

An “enantiomer” is one of two stereoisomers that are mirror images ofeach other, but are non-superposable (not identical).

“Pharmaceutically acceptable carrier” is any excipient which isnon-toxic and aids in a drug's function (see also, Rowe R C et al.,Handbook of Pharmaceutical Excipients, 5^(th) ed., 2006.)

The following examples are provided to further illustrate the presentinvention but, of course, should not be construed as in any way limitingits scope.

All experiments were performed under anhydrous conditions (i.e. drysolvents) in an atmosphere of argon, except where stated, usingoven-dried apparatus and employing standard techniques in handlingair-sensitive materials. Aqueous solutions of sodium bicarbonate(NaHCO3) and sodium chloride (brine) were saturated.

Analytical thin layer chromatography (TLC) was carried out on MerckKiesel gel 60 F254 plates with visualization by ultraviolet and/oranisaldehyde, potassium pennanganate or phosphomolybdic acid dips.

NMR spectra: 1H Nuclear magnetic resonance spectra were recorded at 400MHz. Data are presented as follows: chemical shift, multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, qn=quintet, dd=doublet ofdoublets, m=multiplet, bs=broad singlet), coupling constant (J/Hz) andintegration. Coupling constants were taken and calculated directly fromthe spectra and are uncorrected.

Low resolution mass spectra: Electrospray (ES+) ionization was used. Theprotonated parent ion (M+H) or parent sodium ion (M+Na) or fragment ofhighest mass is quoted. Analytical gradient consisted of 10% ACN inwater ramping up to 100% ACN over 5 minutes unless otherwise stated.

High performance liquid chromatography (HPLC) was use to anaylize thepurity of triazine derivatives. HPLC was performed on a PhenomenexSynergi Polar-RP, 4u, 80A, 150×4.6 mm column using a vShimadzusystemequipted with SPD-M10A Phosphodiode Array Detector.Mobile phase A waswater and mobile phase B was acetonitrile with a gradient from 20% to80% B over 60 minutes and re-equilibrate at A/B (80:20) for 10 minutes.UV detection was at 220 and 54 nm.

Example 1

A 250 mL flask was charged with potassium tert-butoxide (5.75 g, 51.26mmol) and tetrabydrofuran (50 m L). The resulting suspension was cooledto 5-10° C. before ethyl acetoacetate (6.67 g, 51.26 mmol) was I added.The rate of addition was controlled so that the internal temperature ofthe reactor did not exceed 15° C. The resulting mixture becamehomogeneous and was pale yellow in color. After addition was completed,the reaction mixture was cooled to 0° C. and then2,3,4,6-tetrafluoronitrobenzene (5.00 g, 25.63 mol) in tetrabydrofuran(20 m L) was added. After addition was complete, the resulting brownreaction mixture was stirred at about 0° C. (ice batch) for 30 min. 1 NHCl were slowly added and the brown solution eventually became a clearyellow solution. The pH of the aqueous phase was pH 6. The mixture was Iextracted with ethyl acetate (3×) and the combined organic extracts werewashed with brine (50 mL) and concentrated in vacua to afford orangeoil.

The oil obtained above was charged into a 250 L round bottom flask anddissolved in glacial acetic acid (25 mL). Sulfuric acid (cone., 15 mL)was then added and a vigorous evolution of gas was observed in additionto a slight exotherm. Stirring was initiated and the reaction mixturewas heated at 70° C. for 7 h, after which time TLC analysis indicated100% conversion. The reaction mixture was cooled to between 15° C. to20° C. and ethyl acetate (200 mL) was added followed by the addition ofwater (100 mL). Sat. NaOH solution in water was added slowly to adjustthe pH˜7. The mixture was extracted with ethyl acetate (3×100 mL). Thecombined organic extracts were washed with brine. The brown organicextracts were concentrated under reduced pressure to afford crudecompound as brown oil.

The crude product was purified by column chromatography (silica gel,0-30% ethyl acetate in hexanes) to give the desired compound 3.00 g (50%yield, 1). The other component is the isomer (minor product, 1.00 g,16%). 1H NMR (DMSO-d6, 400 MHz) δ 8.01 (ddd, J=17.3 Hz, J=10.4 Hz, J=6.7Hz, 1H), 4.15 (d, J=1.7 Hz, 2H), 2.24 (s, 3H).

Example 2

A mixture of compound 1 (2.82 g, 12.10 mmol) and potassium carbonate(1.83 g, 13.30 mmol) in methanol (50 mL) was heated at 35° C. for 1.5 h.TLC was checked and the starting material was consumed. The reactionmixture was then cooled and concentrated in vacuo to remove most of themethanol. The residue was diluted with ethyl acetate (100 mL) and water.The mixture was neutralized with 2N HCl to PH 4˜5. The mixture wasextracted with ethyl acetate/hexanes (95/5, 3×100 ml). The combinedorganic layer was washed with brine, dried (Na2SO4) and concentrated togive yellow solids (2) 2.90 g (98% yield), which was used directly fromthe next step reaction without purification. 1H NMR (DMSO-d6, 400 MHz) δ7.47 (dd, J=12.7 Hz, J=7.4 Hz, 1H), 4.07 (d, J=1.96 Hz, 2H), 3.95 (s,3H), 2.20 (s, 3H).

Example 3

Method 1: A mixture of1-(25-difluoro-3-methoxy-6-nitrophenyl)-propan-2-one (compound 2) fromprevious step and pyridinium chloride (5 equiv.) was stirred at 175° C.for 120 min. The reaction was cooled to room temperature, diluted I withIN HCl and ethyl acetate and filtered. The filtrate was washed withbrine (2×), dried and concentrated in vacua to give compound 3 of1-(2-fluoro-3-hydroxy-6 nitrophenyl)-propan-2-one as a pink solid, whichwas used without further purification for the next step.

Method 2: A mixture of compound 1 (33.0 g, 141.5 mmol), NaOAc (134.8 g,7.5 eq) and dimethylformamide (450 mL) was stirred at 65° C. for 12 h.The solvent was evaporated under reduced pressure. The crude residue wasdissolved in water (800 mL) and EtOAc (200 mL). The mixture wasextracted with EtOAc (2×400 mL). The organic layer was further washedwith brine (1×150 mL), dried over Na₂SO₄ and concentrated to givecompound 3 (40 g). The crude residue was used in the next step withoutfurther purification.

Example 4

To a solution of sodium dithionite (15.91 g, 91.36 mmol) in water (150mL) was added a solution of compound 2 (2.80 g, 11.42 mmol) in dioxane(17 ml) dropwise at room temperature. After addition, the mixture wasstirred at room temperature until TLC analysis indicated no startingmaterial remained (overnight). Upon completion, the white solids formedwas collected by filtration and washed by water (3×15 ml). the solidswere dried under vacuum overnight to give the desired product 4 as whitesolids (820 mg, 36% yield). The compound was used directly for the nextstep reaction without purification. ¹H NMR (DMSO-d6, 400 MHz) δ11.41(br, 1H), 6.82 (dd, J=12.1 Hz, J=6.2 Hz, 1H), 6.17 (s, 1H), 3.77 (s,3H), 2.34 (s, 3H); ESI-MS: calcd for (C10H9F2NO) 197, found 198 (MH⁺).

Example 5

Method 1: To a cold solution of compound 4 (350 mg, 1.78 mmol) indichlorpmethane (25 ml) was added a solution of boron tribromide (1N inDCM, 6.00 mL, 6.00 mmol) at −78° C. The mixture was slowly warmed up toroom temperature and stirred about 1 h. TLC analysis indicated thecompletion of the reaction. The mixture was poured into ice, sat. NaHCO₃was added. The mixture was extracted with DCM (3×). The organic waswashed with brine, dried (Na₂SO₄) and concentrated to give brown solidsof 5 (281 mg, 86% yield) which was used directly for the next stepreaction without purification. ¹H NMR (DMSO-d6, 400 MHz) δ 11.25 (s,1H), 9.05 (s, 1H), 6.47 (dd, J=11.7 Hz, J=6.4 Hz, 1H), 6.10 (s, 1H),2.32 (s, 3H).

Method 2: Compound 3 (9.09 g, 39.33 mmol) was added to a round bottomflask. Water (200 mL) was added, and the yellow suspension was stirredat RT. Sodium dithionite (53 g, 304.42 mmol) was added in severalportions and the reaction mixture was stirred room temperature until TLCanalysis indicated no starting material remained. Upon completion, thereaction mixture was cooled to 0° C. and the tan solid product wascollected by vacuum filtration. The wet product was dried under highvacuum to afford compound 5 of 4,7-difluoro-2-methyl-1H-indol-5-ol (3.80g) which was characterized by ¹H NMR as in Method 1.

Example 6

To a solution of 4,4-dichloro-2-methlysulfonyl pyrimidine (5.0 g, 25.6mmol) in DMF (20.0 mL) was added a solution of 3-amino-5-cyclopropylpyrazole (3.5 g, 28.2 mmol) and DIPEA (4.9 mL, 28.2 mmol) in DMF (5.0mL) at room temperature. Sodium iodide (4.2 g, 28.2 mmol) was added andreaction was stirred overnight at 60° C. overnight. Then water was addedand solid was collected by filtration. Filtrate was extracted with EtOActwice. Combined organic solvent was dried over sodium sulfate, filteredand concentrated. Residue provide compound 10 as light yellow solid (6.2mg, 96%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.14 (bs, 1H, NH), 10.16 (bs, 1H,NH), 3.31 (s, 1H), 2.46 (s, 3H, CH₃), 1.88-1.84 (m, 1H, CH), 0.92-0.64(m, 4H, Ar—H).

Example 7

To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(methylthio)pyrimidin-4-amine(6.0 g, 23.8 mmol) in MeOH (160.0 mL) was added a solution of oxone(33.7 g, 54.8 mmol) in water (140.0 mL) in portion over 20 minute at 0°C. Reaction was stirred at this temperature for 30 minutes at roomtemperature overnight. Then mixture was filtered and solid wasre-suspended in saturated NaHCO₃ in water. Mixture filtered and solidwas washed with water, diethyl ether. Solid provide compound 11 as lightyellow solid (5.6 g, 75%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.33 (bs, 1H,NH), 10.93 (bs, 1H, NH), 3.34 (s, 3H, CH₃), 1.93-1.89 (m, 1H, CH),0.96-0.68 (m, 4H, Ar—H).

Example 8

The solution of 4, 6-dichloro-2-methylsulfonylpyrimidine (6.87 g, 30.27mmol), and 2-methyl-4-fluro-1-H-indol-5-ol (5.00 g, 30.27 mmol) in THF(100 mL) was cooled to −70° C. with dry-ice/isopropal. A suspension ofpotassium t-butoixde (4.25 g, 37.84 mmole) in THF (50 mL) was added tothe reaction mixture dropwise. The temperature of the mixture wascontrolled below −50° C. After addition, the reaction was stirred at−70° C. for 1.5 h, then warmed up to room temperature over a period of1.5 h. The TLC was checked and both saterting materials were consumed.Saturated ammonium chloride in water was added and the mixture wasextracted with ethyl acetate/hexanes (80/20) three times. The combinedorganic was washed with brine, dried over sodium sulfate andconcentrated. The crude product was a pad of silica gel eluted with 20%ethyl acetate in hexanes. The collected fraction was concentrated togive the desired product as light-yellow solids (QW660) (7.51 g, 79%yield). The solids were directly used for the next step reaction withoutfurther purification.

Example 9

Method 1: (from 7): To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(methylsulfonyl)pyrimidin-4-amine(0.8 g, 2.55 mmol) in tBuOH (50 mL) was added4-fluoro-2-methyl-1H-indol-5-ol (0.46 g, 2.80 mmol) and KOtBu (0.32 g,2.20 mmol) at room temperature. Reaction was stirred at 50° C.overnight. After cooling, reaction was diluted with DCM and washed withsat. NaHCO₃. Aqueous phase was extracted with DCM/isopropanol (4:1)twice. The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacou. The resulting crude product was purified byTeledyne-Isco flash system by using CH₂Cl₂/MeOH, 0 to 5% of methanol indichloromethane to provide compound 12 as light yellow solid (0.86 g,85%).

Method 2: (from 8):

Example 10

To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)pyrimidin-4-amine(100 mg, 0.25 mmol) and 1-methyl piperazine (0.70 mL, 6.25 mmol) inisopropanol (2 mL) was heated to 90° C. overnight in a sealed tube.After cooling, reaction mixture was diluted with dichloromethane andwashed with sat. NaHCO₃. Aqueous phase was extracted withdichloromethane/isopropanol mixture (4:1). The combined organic layerswere dried over anhydrous Na₂SO₄ and concentrated in vacou. Theresulting crude product was purified by Teledyne-Isco flash system byusing CH₂Cl₂/MeOH, 0 to 10% of methanol in dichloromethane to providecompound 10 as light yellow solid (63 mg, 54%). ¹H NMR (400 MHz,DMSO-d₆) δ 11.73 (bs, 1H, NH), 11.21 (bs, H, NH), 9.21 (bs, 1H, NH),7.09-6.08 (m, 4H, Ar—H), 5.25 (bs, 1H, Ar—H), 3.42 (m, 4H, 2CH₂), 2.39(s, 3H, CH₃), 2.35 (m, 4H, 2CH₂), 2.20 (s, 3H, CH₃), 1.49 (m, 1H, CH),0.69-0.13 (m, 4H, Ar—H); ESI-MS: calcd for (C24H27FN8O) 462, found 463[M−H]⁺. HPLC: retention time: 13.47 min. purity: 100%.

Example 11-45

Following the same procedure as in example 10, the compound 11-45 werealso prepared from compound 9 and characterized by LC-MS.

Example number Compounds Structure LC-MS (M + H)⁺ 11

449 12

493 13

450 14

424 15

436 16

464 17

477 18

491 19

463 20

477 21

464 22

466 23

527 24

466 25

526 26

526 27

526 28

507 29

498 30

507 31

477 32

380 33

463 34

563 35

563 36

463 37

463 38

531 39

479 40

515 41

477 42

520 43

479 44

478

Example 45

To a solution ofN-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-(piperazin-1-yl)pyrimidin-4-amine(50 mg, 0.11 mmol) in isopropanol (1 mL) was added 1-fluoro-2-iodoethane(0.02 mL, 0.22 mmol) and K₂CO₃ (76 mg, 055 mmol) and was heated to 75°C. overnight in a sealed tube. After cooling, reaction mixture wasdiluted with dichloromethane and washed with sat. NaHCO₃. Aqueous phasewas extracted with dichloromethane. The combined organic layers weredried over anhydrous Na₂SO₄ and concentrated in vacou. The resultingcrude product was purified by Teledyne-Isco flash system by usingCH₂Cl₂/MeOH, 0 to 10% of methanol in dichloromethane to provide compound45 as light yellow solid (20 mg, 36%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.72(bs, 1H, NH), 11.21 (bs, H, NH), 9.21 (bs, 1H, NH), 7.10-6.04 (m, 4H,Ar—H), 5.25 (bs, 1H, Ar—H), 4.63-2.38 (m, 15H), 1.51 (m, 1H, CH),0.69-0.11 (in, 4H, Ar—H); ESI-MS: calcd for (C25H28F2N8O) 494, found 495[M+H]⁺. HPLC: retention time: 15.02 min. purity: 86%.

Example 46

To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)pyrimidin-4-amine(0.100 g, 0.143 mmol),1-N-boc-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine(0.058 g, 0.188 mmol), and tetrakis(triphenylphosphine)palladium (0)(0.026 g, 0.025 mmol) in dimethoxylethane (2 mL) under argon was addedaqueous 2M Na₂CO₃ (0.276 mL, 0.552 mmol). The mixtures were degassedwith a stream of argon for 3 min, then it was heated to 90° C. for 24 hin a sealed tube. The cooled mixtures were quenched with 1N NaOH andextracted with dichloromethane/isopropanol (8:2). The combined organiclayers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. Theresidue was purified by flash chromatography over silica gel withCH₂Cl₂:MeOH (9:1) to give compound 46 (0.036 g, 53%) as a light yellowsolid. ¹H NMR (400 MHz, DMSO-d): δ 11.85 (bs, 1H), 11.24 (s, 1H), 9.92(bs, 1H), 7.10 (d, 1H, J=8.4 Hz), 6.86 (m, 1H), 6.76 (bs, 1H), 6.53 (bs,1H), 6.18 (s, 1H), 5.25 (bs, 1H), 4.02 (m, 2H), 3.50 (m, 2H), 2.38 (bs,5H), 1.41 (bs, 10H), 0.65 (m, 2H),−0.02 (m, 2H). MS (ESI): Calcd. forC₂₉H₃₂FN₇O₃: 545, found 546 (M+H).

Example 47

Compound 46 (0.035 g, 0.062 mmol) was dissolved with 20% trifluoromethylacetic acid in dichloromethane (5 mL) and stirred for 3 h. Then themixture was neutralized with 1N aq. NaOH and extracted withdichloromethane/isopropanol (8:2) mixture. The combined organic layerswere dried over anhydrous Na₂SO₄ and concentrated in vacuo to give 47(0.038 g, 59%) as a light yellow solid. ¹H NMR (400 MHz, DMSO-d): δ11.84 (bs, 1H), 11.23 (s, 1H), 9.84 (bs, 1H), 7.09 (d, 1H, J=8.4 Hz),6.86 (m, 1H), 6.80 (bs, 1H), 6.51 (bs, 1H), 6.18 (s, 1H), 5.27 (bs, 1H),3.37 (m, 2H), 2.87 (m, 2H), 2.38 (s, 3H), 2.24 (m, 2H), 1.44 (m, 1H),1.22 (m, 1H), 1.02 (d, 1H, J=6.0 Hz), 0.65 (m, 2H), 0.01 (m, 2H). MS(ESI): Calcd. for C₂₄H₂₄FN₇O: 445, found 446 (M+H).

Example 48

N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-(1,2,3,6-tetrahydropyridin-4-yl)pyrimidin-4-amine(0.040 g, 0.090 mmol), 1-bromo-2-fluoroethane (0.031 g, 0.180 mmol), andK₂CO₃ (0.062 g, 0.449 mmol) dissolved with THF/CH₃CN (4 mL/4 mL) solventmixtures. The sealed tube was stirred 20 h at 75° C. and after coolingthe solvent was removed to minimum. The crude was partitioned betweenDCM/isopropanol (8:2) and water. The organic layers were washed withsat. NaHCO₃, dried over anhydrous Na₂SO₄, and concentrated in vacou. Theresidue was purified by flash chromatography over silica gel withCH₂Cl₂:MeOH (9:1) to give compound 48 (0.036 g, 83%) as a light yellowsolid. ¹H NMR (400 MHz, DMSO-d): δ 11.83 (bs, 1H), 11.24 (s, 1H), 9.87(bs, 1H), 7.10 (d, 1H, J=8.4 Hz), 6.86 (m, 1H), 6.75 (bs, 1H), 6.18 (s,1H), 5.24 (bs, 1H), 4.63 (m, 1H), 4.53 (m, 1H), 3.46 (m, 1H), 3.40 (m,1H), 3.21 (m, 2H), 2.78 (m, 1H), 2.71 (m, 3H), 2.37 (m, 5H), 1.43 (m,1H), 0.65 (m, 2H), −0.03 (m, 2H). MS (ESI): Calcd. for C₂₆H₂₇F₂N₇O: 491,found 492 (M+H).

Example 49

To a solution ofN-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-(1,2,3,6-tetrahydropyridin-4-yl)pyrimidin-4-amine(0.025 g, 0.056 mmol) in tetrahydrofuran (10 mL) was added formaldehyde(37% in water, 0.911 g, 0.112 mmol) and stirred for 10 min. Thenfollowed by the addition of sodium triacetoxyborohydride (0.024 g, 0.112mmol) and continued to for 20 h. The mixtures were quenched with sat.NaHCO₃ and extracted with dichloromethane/isopropanol (8:2) mixtures.The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified by flash chromatographyover silica gel with CH₂Cl₂:MeOH (8:2) to give compound 50 (0.025 g,96%) as a light yellow solid. ¹H NMR (400 MHz, DMSO-d): δ 11.82 (bs,1H), 11.23 (s, 1H), 9.85 (bs, 1H), 7.09 (d, 1H, J=8.4 Hz), 6.84 (m, 1H),6.75 (m, 1H), 6.18 (s, 1H), 5.23 (m, 1H), 4.06 (m, 1H), 3.15 (m, 3H),3.05 (m, 2H), 2.55 (m 2H), 2.37 (m, 5H), 2.27 (s, 3H). MS (ESI): Calcd.for C₂₅H₂₆FN₇O: 459, found 460 (M+H).

Example 50

To a suspension of 4,6-dichloro-2-methylsulfanyl-pyrimidin (390 m g,2.00 mmol) in DMF (3.0 mL) was added a solution of(E)-5-(prop-1-en-1-yl)-1H-pyrazol-3-amine (271 mg, 2.20 mmol) and DIPEA(0.42 mL, 2.40 mmol) in DMF (2.0 mL) at room temperature, followed byaddition of sodium iodide (330 mg, 2.20 mmol). After addition, themixture was stirred at 50° C. for overnight. TLC was checked and thestarting material was consumed. The mixture was poured in to water (˜50mL) and the solids were collected by filtration, washed with water,hexane. The desired product (50) was obtained as yellow solids afterdring on vac line (286 mg, 51% yield). The product was used directly forthe next step reaction without purification.

Example 51

To a cold solution of compound 50 (280 m g, 1.00 mmol) in methanole (6mL) was added a suspension of oxane (1500 mg, 2.44 mmol) at 0° C. Afteraddition, the mixture was stirred at 0° C. ° C. for 0.5 hours, then roomtemperature for 1 hour. TLC was checked and the starting material wasconsumed. Water was added to the reaction mixture. The mixture wasextracted with ethyl acetate three times. The combined organic waswashed with brine, dried over sodium sulfate and concentrated. The crudeproduct was purified on column (0-5% methanol in DCM). The collectedfraction was concentrated to give the desired product as off-whitesolids (51) (30 mg, 9.5% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 12.55 (br,1H), 10.98 (br, 1H), 8.00-6.00 (m, 4H), 3.38 (s, 3H), 1.90 (br, 3H);ESI-MS: calcd for (C11H12C1N5O2S) 313, found 314 (MH⁺).

Example 52

Method 1: To a suspension of6-chloro-N-(5propenyl-1H-pyrazol-3-yl)-2-(methylsulfonyl)pyrimidin-4-amine(51) (200 m g, 0.63 mmol) and 2-methyl-4-fluro-1-H-indol-5-ol (110 mg,0.67 mmol) in t-BuOH (15.0 mL) was added t-BuOK (79 mg, 0.70 mmol) atroom temperature. After addition, the mixture was stirred at 55° C. for16 hours. TLC was checked and the starting material was consumed. Waterwas added to the reaction mixture. The mixture was extracted withDCM/i-propol (90/10) three times. The combined organic was washed withbrine, dried over sodium sulfate and concentrated. The crude product waspurified on column (0-10% methanol in DCM). The collected fraction wasconcentrated to give the desired product as pink solids (52) (163 mg,64% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 12.15 (br, 1H), 11.35 (br, 1H),10.35 (br, 1H), 7.14 (d, J=8.8 Hz, 1H), 6.90 (t, J=8.0 Hz, 1H), 6.40(br, 1H), 6.22 (s, 1H), 5.70-5.10 (m, 3H), 2.40 (s, 3H), 1.78 (br, 3H);ESI-MS: calcd for (C19H16ClFN6O) 398, found 399 (MH⁺).

Method 2: The solution of compound 8 (5.00 g, 16.02 mmol),(E)-5-(prop-1-en-1-yl)-1H-pyrazol-3-amine (3.45 g, 28.03 mmol), sodiumiodide (3.60 g, 24.03 mmol) and DIPEA (4.20 ml, 24.03 mmol) in DMF (50mL) was stirred at 65° C. for 48 hours TLC was checked and the startingmaterial was consumed. The mixture was poured into water (500 ml) andcooled with ice. The solids were collected by filtration, washed bywater and hexanes. The slides were dissolved into dichlomethane/meanol.The solution was concentrated to minimum amount of solvents. The solidswere collected by filtration, washed by methanol to give yellow solids(52) (3.88 g, 61% yield)). The mothe liquid was recovered. ¹H NMR (400MHz, DMSO-d₆). ¹H NMR (400 MHz, DMSO-d₆) δ 12.15 (br, 1H), 11.35 (br,1H), 10.35 (br, 1H), 7.14 (d, J=8.8 Hz, 1H), 6.90 (t, J=8.0 Hz, 1H),6.40 (br, 1H), 6.22 (s, 1H), 5.70-5.10 (m, 3H), 2.40 (s, 3H), 1.78 (br,3H); ESI-MS: calcd for (C19H16ClFN6O) 398, found 399 (MH⁺).

Example 53

The solution of compound 52 (1.50 g, 3.76 mmol), 1-methylpiperazine(2.83 g, 28.21 mmol) and DIPEA (1.64 ml, 9.40 mmol) in iso-propal (20.0mL) and acetonitrile (5.0 mL) was stirred at 85° C. for 2 days. TLC waschecked and the starting material was consumed. The reaction mixture wasconcentrated and water was added. The mixture was extracted withDCM/isopropal (10/1) three times. The combined organic was washed with,sodium bicarbonate, brine, dried over sodium sulfate and concentrated.The crude product was purified crytalization from MeOH (˜10 mL). Thelight-yellow solids were collected by filtration, washed with cold MeOH(1×) to give compound 53 (1.10 g, 63% yield). ¹H NMR (400 MHz, DMSO-d₆)δ 11.80 (br, 1H), 11.19 (br, 1H), 9.30 (br, 1H), 7.03 (d, J=8.8 Hz, 1H),6.78 (t, J=8.0 Hz, 1H), 6.14 (s, 1H), 5.80-5.60 (m, 2H), 5.47 (s, 1H),5.20 (br, 1H), 3.40 (br, 4H), 2.43 (3H, obs with solvent peak), 2.34 (s,3H), 2.27 (br, 4H), 1.65 (d, J=6.0 Hz, 3H); ESI-MS: calcd for(C24H27FN8O) 462, found 463 (MH⁺).

Example 54-73

Following the same procedure as in example 53, the compound 54-73 werealso prepared from compound 52 and characterized by LC-MS.

Example number Compounds Structure LC-MS (M + H)⁺ 54

507 55

527 56

491 57

498 58

478 59

464 60

464 61

450 62

505 63

477 64

478 65

531 66

493 67

450 68

449 69

394 70

420 71

434 72

448 73

408

Example 74

The solution of compound 68 (50 m g, 0.11 mmol), 1-fluoro-2-iodoethane(40.72 mg, 0.23 mmol) and potassium carbonate (77 mg, 0.56 mmol) inAcetonitrile/THF (3.0 mL/3.0 mL) was stirred at 75° C. for 3 days. TLCwas checked and the starting material was consumed. The solvents wereremoved and water was added. The mixture was extracted withDCM/isopropal (9/1) three times. The combined organic was washed withbrine, dried over sodium sulfate and concentrated. The crude product waspurified on column (0-10% methanol in DCM). The collected fraction wasconcentrated to give the desired product as off-white solids (74) (25mg, 45% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (br, 1H), 11.24 (br,1H), 9.30 (br, 1H), 7.09 (d, J=8.8 Hz, 1H), 6.83 (t, J=8.0 Hz, 1H), 6.19(s, 1H), 5.80-5.00 (m, 4H), 4.49 (m, 2H), 3.40 (br, 4H), 2.69 (m, 2H),2.38 (m, 4H), 1.68 (d, J=6.8 Hz, 3H), 1.10 (s, 3H); ESI-MS: calcd for(C25H28F2N8O) 494, found 495 (MH⁺).

Example 75

To a solution of(E)-6-chloro-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-N-(5-(prop-1-en-1-yl)-1H-pyrazol-3-yl)pyrimidin-4-amine(0.075 g, 0.188 mmol),1-N-boc-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine(0.093 g, 0.301 mmol), and tetrakis(triphenylphosphine)palladium (0)(0.019 g, 0.019 mmol) in dimethoxylethane (4 mL) under argon was addedaqueous 2M Na₂CO₃ (0.206 mL, 0.414 mmol). The mixtures were degassedwith a stream of argon for 3 min then it was heated to 90° C. for 24 hin a sealed tube. The cooled mixtures were quenched with 1N NaOH andextracted with dichloromethane/isopropanol (8:2). The combined organiclayers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. Theresidue was then dissolved with 20% trifluoromethyl acetic acid indichloromethane (5 mL) and stirred for 3 h. The reaction was neutralizedwith 1N aq. NaOH and extracted with dichloromethane/isopropanol (8:2)mixture. The combined organic layers were dried over anhydrous Na₂SO₄and concentrated in vacuo to give 75 (0.036 g, 44%) as a yellow solid.¹H NMR (400 MHz, DMSO-d): δ 12.02 (bs, 1H), 11.29 (s, 1H), 9.98 (bs,1H), 7.11 (d, 1H, J=8.4 Hz), 6.87 (m, 1H), 6.83 (bs, 1H), 6.52 (bs, 1H),6.20 (s, 1H), 5.66 (m, 1H), 5.56 (bs, 1H), 5.25 (m, 1H), 3.50 (s, 1H),3.43 (m, 2H), 2.92 (m, 2H), 2.41 (s, 3H), 2.28 (m, 2H), 1.69 (dd, 3H,J=5.2, 1.2 Hz). MS (ESI): Calcd. for C₂₄H₂₄FN₇O: 445, found 446 (M+H).

Example 76

The solution of5-((4,6-dichloropyrimidin-2-yl)oxy)-4-fluoro-2-methyl-1H-indole (0.5 g,1.60 mmol), 5-methylthiazol-2-amine (0.22 g, 1.92 mmol), sodium iodide(0.29 g, 1.92 mmol) and DIPEA (0.39 mL, 1.92 mmol) in DMF (16.0 mL) wasstirred at 70° C. overnight. The mixture was slowly added to theice-water (10.0 mL). The mixture was cooled by ice bath and the solidswere collected by filtration, washed with water and hexanes to providecompound 77 as pale white solid (0.59 g, 95%). ¹H NMR (400 MHz, DMSO-d₆)δ 11.34 (bs, 1H), 9.81 (bs, H), 8.70 (bs, 1H), 7.22 (m, 1H), 7.14 (m,1H), 6.97 (m, 1H), 6.25 (m, 1H), 2.40 (s, 3H), 2.02 (s, 3H); ESI-MS:calcd for (C17H13ClFN5OS) 389, found 390 [M−H]⁺.

Example 77

A mixture ofN-(6-chloro-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)pyrimidin-4-yl)-5-methylthiazol-2-amine(0.1 g, 0.26 mmol), 1-methyl piperazine (32 mg, 1.25 mmol), Pd(OAc)₂(8.2 mg, 0.036 mmol) and K₂CO₃ (0.57 g, 4.10 mmol) in THF (1.5 mL) andDMF (1.0 mL) were heated in the microwave for 1.5 h at 60° C. Thereaction mixture was cooled down and water was added. The resultingmixture was extracted with EtOAc and the combined extracts were washedwith brine, dried over anhydrous Na₂SO₄ and then concentrated underreduced pressure. The resulting crude product was purified byTeledyne-Isco flash system by using CH₂Cl₂/MeOH, 0 to 20% of methanol indichloromethane to provide compound 77 as off brown solid (20 mg, 17%);¹H NMR (400 MHz, DMSO-d₆) δ 11.27 (bs, 1H), 9.48 (bs, H), 7.35 (bs, 1H),7.15-7.00 (m, 2H), 6.85 (m, 1H), 6.22 (m, 1H), 3.45 (m, 4H), 2.41 (s,3H), 2.32 (m, 4H), 2.18 (s, 3H), 1.95 (s, 3H); MS (ESI): Calcd. forC22H24FN7OS: 453, found 454, (M−H)⁺.

Example 78

Following the same procedure as in example 77, the compound 78 was alsoprepared from compound 76 and characterized by LC-MS 441 (M−H)⁺.

Example 79

The solution of 4, 6-dichloro-2-methylsulfonylpyrimidine (3.72 g, 16.38mmol), and 2-methyl-4,7-di-fluro-1-H-indol-5-ol (3.00 g, 16.38 mmol) inTHF (100 mL) was cooled to −78° C. with dry-ice/acetone. A suspension ofpotassium t-butoixde (2.30 g, 20.47 mmole) in THF (50 mL) was added tothe reaction mixture dropwise. The temperature of the mixture wascontrolled below −50° C. After addition, the reaction was stirred at−78° C. for 1 h, then warmed up to room temperature over a period of 1h. The TLC was checked and both saterting materials were consumed.Saturated ammonium chloride in water was added and the mixture wasextracted with ethyl acetate/hexanes (80/20) three times. The combinedorganic was washed with brine, dried over sodium sulfate andconcentrated. The crude product was a pad of silica gel eluted with 15%ethyl acetate in hexanes. The collected fraction was concentrated togive the desired product as light-yellow solids (79) (4.20 g, 78%yield). The solids were directly used for the next step reaction withoutfurther purification.

Example 80

To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(methylsulfonyl)pyrimidin-4-amine(0.7 g, 2.23 mmol) in tBuOH (50 mL) was added4,7-difluoro-2-methyl-1H-indol-5-ol (0.45 g, 2.45 mmol) and KOtBu (0.28g, 2.45 mmol) at room temperature. Reaction was stirred at 50° C. fortwo days. After cooling, reaction was diluted with DCM and washed withsat. NaHCO₃. Aqueous phase was extracted with DCM/isopropanol (4:1)twice. The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacou. The resulting crude product was purified byTeledyne-Isco flash system by using CH₂Cl₂/MeOH, 0 to 5% of methanol indichloromethane to provide compound 80 as light yellow solid (0.49 g,53%).

Compound 80 can also be prepared by reaction of compound 79 with3-cyclopropyl-1H-pyrazol-5-amine with the same procedure as described inexperiment 53 (Method 2).

Example 81

To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4,7-difluoro-2-methyl-1H-indol-5-yl)oxy)pyrimidin-4-amine(80 mg, 0.20 mmol) and 1-methyl piperazine (0.56 mL, 5.00 mmol) inisopropanol (1 mL) was heated to 90° C. overnight in a sealed tube.After cooling, reaction mixture was diluted with dichloromethane andwashed with sat. NaHCO₃. Aqueous phase was extracted withdichloromethane/isopropanol mixture (4:1). The combined organic layerswere dried over anhydrous Na₂SO₄ and concentrated in vacou. Theresulting crude product was purified by Teledyne-Isco flash system byusing CH₂Cl₂/MeOH, 0 to 10% of methanol in dichloromethane to providecompound 81 as light yellow solid (33 mg, 36%). %). ¹H NMR (400 MHz,DMSO-d₆) δ 11.76 (bs, 1H, NH), 11.70 (bs, H, NH), 9.26 (bs, 1H, NH),6.86-5.97 (m, 3H, Ar—H), 5.26 (bs, 1H, Ar—H), 3.43 (m, 4H, 2CH₂), 2.40(s, 3H, CH₃), 2.35 (m, 4H, 2CH₂), 2.20 (s, 3H, CH₃), 1.49 (m, 1H, CH),0.71-0.09 (m, 4H, Ar—H); ESI-MS: calcd for (C24H26F2N8O) 480, found 481[M−H]⁺. HPLC: retention time: 14.84 min. purity: 100%.

Example 82-85

Following the same procedure as in example 81, the compound 82-85 werealso prepared from compound 80 and characterized by LC-MS.

Example number Compounds Structure LC-MS (M + H)⁺ 82

467 83

511 84

468 85

495

Example 86

The solution of compound 79 (4.20 g, 12.72 mmol),(E)-5-(prop-1-en-1-yl)-1H-pyrazol-3-amine (2.82 g, 22.90 mmol), sodiumiodide (2.86 g, 19.08 mmol) and DIPEA (3.33 ml, 19.08 mmol) in DMF (35mL) was stirred at 65° C. for 48 hours TLC was checked and the startingmaterial was consumed. The mixture was cooled with ice and the solidswere collected by filtration, washed by water and hexanes. The slideswere dissolved into dichlomethane/meanol. The solution was concentratedto minimum amount of solvents. The solids were collected by filtration,washed by methanol to give yellow solids (1.90 g). The mothe liquid waspurified by column. The desired parts were collected, concentrated andfiltered to give light-yellow solids (0.82 g). The combined solids (86)were used for the next step reactions (2.72 g, 51%)¹H NMR (400 MHz,DMSO-d₆) δ 12.15 (br, 1H), 11.80 (br, 1H), 10.40 (br, 1H), 6.95 (dd,J=10.8 Hz, J=5.6 Hz, 1H), 6.40 (br, 1H), 6.22 (s, 1H), 5.70-5.10 (m,3H), 2.40 (s, 3H), 1.78 (br, 3H); ESI-MS: calcd for (C19H15CIF2N6O) 416,found 417 (MH⁺).

Alternatively, compound 86 can also be prepared from the reaction of6-chloro-N-(5propenyl-1H-pyrazol-3-yl)-2-(methylsulfonyl)pyrimidin-4-amineand 2-methyl-4,7-di-fluro-1-H-indol-5-ol with the same protocol asdescribed earlier.

Example 87

The solution of 86 (45 m g, 0.11 mmol), 1-methyl piperazine (270 mg,2.70 mmol) and DIPEA (0.10 ml, 0.54 mmol) in iso-propal (3.0 mL) andacetonitrile (1.0 mL) was stirred at 85° C. for 3 days. TLC was checkedand the starting material was consumed. Dilute sodium bicarbonate wasadded and the mixture was extracted with DCM three times. The combinedorganic was washed with brine, dried over sodium sulfate andconcentrated. The crude product was purified on column (0-15% methanolin DCM). The collected fraction was concentrated to give the desiredproduct as off-white solids (87) (33 mg, 66% yield). ¹H NMR (400 MHz,DMSO-d₆) δ 11.90 (br, 1H), 11.71 (br, 1H), 9.30 (br, 1H), 6.85 (dd,J=10.8 Hz, J=5.6 Hz, 1H), 6.29 (s, 1H), 5.80-5.00 (m, 4H), 3.40 (br,4H), 2.40 (m, 7H), 2.18 (s, 3H), 1.68 (d, J=6.8 Hz, 3H); ESI-MS: calcdfor (C24H26F2N8O) 480, found 481 (MH⁺).

Example 88-99

Following the same procedure as in example 87, the compound 88-99 werealso prepared from compound 86 and characterized by LC-MS.

Example number Compounds Structure LC-MS (M + H)⁺ 88

511 89

468 90

467 91

495 92

468 93

482 94

523 95

482 96

496 97

509 98

545 99

495

Example 100

To a solution of(E)-6-chloro-2-((4,7-difluoro-2-methyl-1H-indol-5-yl)oxy)-N-(5-(prop-1-en-1-yl)-1H-pyrazol-3-yl)pyrimidin-4-amine(0.075 g, 0.179 mmol),1-N-boc-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine(0.089 g, 0.287 mmol), and tetrakis(triphenylphosphine)palladium (0)(0.018 g, 0.018 mmol) in dimethoxylethane (4 mL) under argon was addedaqueous 2M Na₂CO₃ (0.197 mL, 0.396 mmol). The mixtures were degassedwith a stream of argon for 3 min then it was heated to 90° C. for 24 hin a sealed tube. The cooled mixtures were quenched with 1N NaOH andextracted with dichloromethane/isopropanol (8:2). The combined organiclayers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. Theresidue was then dissolved with 20% trifluoromethyl acetic acid indichloromethane (5 mL) and stirred for 3 h. The reaction was neutralizedwith 1N aq. NaOH and extracted with dichloromethane/isopropanol (8:2)mixture. The combined organic layers were dried over anhydrous Na₂SO₄and concentrated in vacuo to give 100 (0.041 g, 49%) as a yellow solid.¹H NMR (400 MHz, DMSO-d): δ 12.08 (bs, 1H), 11.77 (s, 1H), 10.04 (bs,1H), 6.89 (q, 1H, J=5.6 Hz), 6.84 (m, 1H), 6.52 (bs, 1H), 6.32 (s, 1H),5.70 (m, 1H), 5.59 (bs, 1H), 5.26 (m, 1H), 3.49 (s, 1H), 3.45 (m, 2H),2.94 (m, 2H), 2.42 (s, 3H), 2.30 (m, 2H), 1.71 (dd, 3H, J=5.2, 1.2 Hz).MS (ESI): Calcd. for C₂₄H₂₃F₂N₇O: 463, found 464 (M+H).

Example 101

To a solution of(E)-2-((4,7-difluoro-2-methyl-1H-indol-5-yl)oxy)-N-(5-(prop-1-en-1-yl)-1H-pyrazol-3-yl)-6-(1,2,3,6-tetrahydropyridin-4-yl)pyrimidin-4-amine(0.022 g, 0.048 mmol) in tetrahydrofuran (10 mL) was added formaldehyde(37% in water, 0.012 g, 0.142 mmol) and stirred for 10 min. Thenfollowed by the addition of sodium triacetoxyborohydride (0.030 g, 0.142mmol) and continued to for 20 h. The mixtures were quenched with sat.NaHCO₃ and extracted with dichloromethane/isopropanol (8:2) mixtures.The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified by flash chromatographyover silica gel with CH₂Cl₂:MeOH (8:2) to give 101 (0.020 g, 87%) as alight yellow solid. ¹H NMR (400 MHz, DMSO-d): δ 12.08 (bs, 1H), 11.77(s, 1H), 10.03 (bs, 1H), 7.09 (m, 1H), 6.79 (m, 1H), 6.52 (m, 1H), 6.32(s, 1H), 5.67 (m, 1H), 5.57 (m, 1H), 5.24 (m, 1H), 3.06 (m, 2H), 2.56(m, 2H), 2.42 (s, 3H), 2.40 (m, 2H0, 2.28 (s, 3H), 1.71 (dd, 3H, J=6.8,1.2 Hz). MS (ESI): Calcd. for C₂₅H₂₅F₂N₇O: 477, found 478 (M+H).

Example 102

To a suspension of6-chloro-N-(5-methyl-1H-pyrazol-3-yl)-2-(methylsulfonyl)pyrimidin-4-amine(700 in g, 2.43 mmol) and 2-methyl-4,7-di-fluro-1-H-indol-5-ol (499 mg,2.72 mmol) in t-BuOH (50.0 mL) was added t-BuOK (33 mg, 2.92 mmol) atroom temperature. After addition, the mixture was stirred at 55° C. for16 hours. TLC was checked and the starting material was consumed. Waterwas added to the reaction mixture. The mixture was extracted withDCM/i-propol (90/10) three times. The combined organic was washed withbrine, dried over sodium sulfate and concentrated. The crude product waspurified on column (0-10% methanol in DCM). The collected fraction wasconcentrated to give the desired product as pink solids (102) (435 mg,46% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (br, 1H), 11.77 (br, 1H),10.30 (br, 1H), 6.95 (dd, J=10.8 Hz, J=5.6 Hz, 1H), 6.40 (br, 1H), 6.32(s, 1H), 5.25 (br, 1H), 2.40 (s, 3H), 1.78 (s, 3H); ESI-MS: calcd for(C17H13CIF2N6O) 390, found 391 (MH⁺).

Example 103

The solution of compound 102 (50 m g, 0.13 mmol), 1-ethyl piperazine(365 mg, 3.20 mmol) and DIPEA (0.11 ml, 0.64 mmol) in iso-propal (2.5mL) was stirred at 85° C. for 3 days. TLC was checked and the startingmaterial was consumed. Dilute sodium bicarbonate was added and themixture was extracted with DCM three times. The combined organic waswashed with brine, dried over sodium sulfate and concentrated. The crudeproduct was purified on column (0-15% methanol in DCM). The collectedfraction was concentrated to give the desired product as off-whitesolids (103) (45 mg, 75% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.65 (br,2H), 9.20 (br, 1H), 6.82 (dd, J=10.8 Hz, J=5.6 Hz, 1H), 6.27 (s, 1H),6.07 (br, 1H), 5.35 (s, 1H), 3.40 (br, 4H), 2.31 (m, 9H), 1.95 (s, 3H),1.00 (t, J=7.2 Hz, 3H); ESI-MS: calcd for (C23H26F2N8O) 468, found 469(MH⁺).

Example 104-109

Following the same procedure as in example 103, the compound 104-109were also prepared from compound 102 and characterized by LC-MS.

Example number Compounds Structure LC-MS (M + H)⁺ 104

455 105

485 106

441 107

442 108

455 109

458

Example 110

The solution of 4, 6-dichloro-2-methylsulfonylpyrimidine (938 mg, 4.13mmol), and 2-methyl-4-chloro-1-H-indol-5-ol (750 mg, 4.13 mmol) in THF(20 mL) was cooled to −78° C. with dry-ice/acetone. A suspension ofpotassium t-butoixde (510 mg, 4.54 mmol) in THF (10 mL) was added to thereaction mixture dropwise. The temperature of the mixture was controlledbelow −50° C. After addition, the reaction was stirred at −78° C. for 1h, then warmed up to room temperature over a period of 1.5 h. The TLCwas checked and both saterting materials were consumed. Saturatedammonium chloride in water was added and the mixture was extracted withethyl acetate/hexanes (95/5) three times. The combined organic waswashed with brine, dried over sodium sulfate and concentrated. The crudeproduct was a pad of silica gel eluted with 20% ethyl acetate inhexanes. The collected fraction was concentrated to give the desiredproduct as light-yellow solids (110) (900 mg, 66% yield). The solidswere directly used for the next step reaction without furtherpurification. ¹H NMR (400 MHz, DMSO-d₆) δ 11.42 (s, 1H), 7.75 (s, 1H),7.30 (d, J=8.4 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.21 (s, 1H), 2.41 (s,3H); ESI-MS: calcd for (C13H8C13N3O) 326, found 327 (MH⁺).

Example 111

The solution of5-((4,6-dichloropyrimidin-2-yl)oxy)-4-chloro-2-methyl-1H-indole (0.85 g,2.59 mmol), (E)-5-(prop-1-en-1-yl)-1H-pyrazol-3-amine (0.48 g, 3.89mmol), sodium iodide (0.58 g, 3.89 mmol) and DIPEA (0.68 mL, 3.89 mmol)in DMF (10.0 mL) was stirred at 70° C. for 2 days. The mixture wasslowly added to the water (200.0 mL). The mixture was cooled by ice bathand the solids were collected by filtration, washed by water andhexanes. The solid was dissolved in CH₂Cl₂/MeOH and concentrated.Resulting crude was purified by Teledyne-Isco flash system by usingCH₂Cl₂/MeOH, 0 to 5% of methanol in dichloromethane to provide compound111 as light yellow solid (? g, ?%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.13(bs, 1H, NH), 11.42 (bs, H, NH), 10.38 (bs, 1H, NH), 7.32-5.17 (m, 7H,5Ar—H, 2CH), 2.44 (s, 3H, CH₃), 1.71 (d, 3H, CH₃); ESI-MS: calcd for(C19H16C12N6O) 415, found 416 [M+H]⁺.

Example 112

Solution of(E)-6-chloro-2-((4-chloro-2-methyl-1H-indol-5-yl)oxy)-N-(5-(prop-1-en-1-yl)-1H-pyrazol-3-yl)pyrimidin-4-amine(50 mg, 0.12 mmol) and 1-methyl piperazine (0.067 mL, 0.60 mmol) inisopropanol (1.0 mL) was heated to 85° C. overnight. After cooling,reaction mixture was diluted with dichloromethane and washed with water.Aqueous phase was extracted with dichloromethane. The combined organiclayers were dried over anhydrous Na₂SO₄ and concentrated. The resultingcrude product was purified by Teledyne-Isco flash system by usingCH₂Cl₂/MeOH, 0 to 10% of methanol in dichloromethane to provide compound112 as white solid (25 mg, 43%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (bs,1H, NH), 11.31 (bs, H, NH), 9.32 (bs, 1H, NH), 7.27-5.34 (m, 7H, 5Ar—H,2CH), 3.44 (m, 4H, 2CH₂), 2.43-2.35 (m, 7H, 2CH₂, CH₃), 2.21 (s, 3H,CH₃), 1.71 (d, 3H, CH₃); ESI-MS: calcd for (C24H27C1N8O) 478, found 479[M+H]⁺. HPLC: retention time: 15.70 min. purity: 92%.

Example 113-116

Following the same procedure as in example 112, the compound 113-116were also prepared from compound 111 and characterized by LC-MS.

Example number Compounds Structure LC-MS (M + H)⁺ 113

466 114

465 115

494 116

510

Example 117

To a cold (0° C.) suspension of sodium hydride (60%, 1.54 g, 38.5 mmol)in DMF (20 mL) was added slowly a solution of5-((4,6-dichloropyrimidin-2-yl)oxy)-4-fluoro-2-methyl-1H-indole(compound 8, 6.00 g, 19.2 mmol) and idomethan (5.46 g, 38.5 mmol) in DMF(28 mL). Thereaction mixture was stirred at 0° C. for 2 hours. TLC waschecked and the starting material was consumed. The mixture was pouredto cold water potionwise and the container was put to ice bath. Thewhite solids were collected by filtration, washed with water andtrinuated with hexanes. After further dring on vacuum line, 6.03 g whitesolids (117) were obtained (96% yield) No further purification wasconducted. ¹H NMR (400 MHz, DMSO-d₆) δ 7.76 (s, 1H), 7.26 (d, J=8.8 Hz,1H), 7.03 (t, J=8.0 Hz, 1H) 6.34 (s, 1H), 3.69 (s, 3H), 2.41 (s, 3H);ESI-MS: calcd for (C14H10Cl2FN3O) 325, found 326 (MH⁺).

Example 118

The solution of5-((4,6-dichloropyrimidin-2-yl)oxy)-4-fluoro-1,2-dimethyl-1H-indole(compound 117, 2.68 g, 8.22 mmol),(E)-5-(prop-1-en-1-yl)-1H-pyrazol-3-amine (1.77 g, 14.38 mmol), sodiumiodide (1.85 g, 12.33 mmol) and DIPEA (2.15 ml, 12.33 mmol) in DMF (80mL) was stirred at 65° C. for 24 hours TLC was checked and the startingmaterial was consumed. The mixture was poured into water (500 mL) andcooled with ice. The solids were collected by filtration, washed bywater and hexanes. The slides were dissolved into dichlomethane/meanol.The solution was concentrated to minimum amount of solvents. The solidswew collected by filtration, washed by methanol to give yellow solids(118) (2.27 g, 67% yield)). The mothe liquid was recovered. ¹H NMR (400MHz, DMSO-d₆). ¹H NMR (400 MHz, DMSO-d₆) δ 12.15 (br, 1H), 10.35 (br,1H), 7.14 (m, 1H), 6.90 (m, 1H), 6.40 (br, 1H), 6.22 (s, 1H), 5.70-5.10(m, 3H), 3.80 (br, 3H) 2.40 (s, 3H), 1.78 (br, 3H); ESI-MS: calcd for(C20H18ClFN6O) 412, found 413 (MH⁺).

Example 119

With same procedure as described in example 120, under the samecondition, the reaction of compound 119 and3-cyclopropylpyrozole-5-amine generated product 119. ¹H NMR (400 MHz,DMSO-d₆). ¹H NMR (400 MHz, DMSO-d₆) δ 12.15 (br, 1H), 10.35 (br, 1H),7.14 (m, 1H), 7.00 (m, 1H), 6.50 (br, 1H), 6.22 (s, 1H), 5.00 (br, 1H),3.80 (s, 3H) 2.40 (s, 3H), 1.50 (br, 1H), 1.20 (br, 2H), 0.80 (br, 2H);ESI-MS: calcd for (C20H18ClFN6O) 412, found 413 (MH⁺).

Example 120

The solution of compound 118 (150 mg, 0.36 mmol), 1-methylpiperazine(181 mg, 1.81 mmol) and DIPEA (0.13 ml, 0.73 mmol) in DMSO (3.5 mL) wasstirred at 75° C. for 3 days. TLC was checked and the starting materialwas consumed. The reaction mixture was poured into dilute sodiumbicarbonate in water (˜1%) and cooled with ice bath. The solids werecollected by filtration, washed by water and hexanes to give light-brownsolids 161 mg. The crude product was trinuated with MeOH, filtered andwashed by MeOH to give compound 120 as light-yellow solids (82 mg, 47%yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (br, 1H), 9.31 (br, 1H), 7.20(d, J=8.8 Hz, 1H), 6.92 (t, J=8.0 Hz, 1H), 6.28 (s, 1H), 5.80-5.60 (m,4H), 3.70 (s, 3H), 3.42 (br, 4H), 2.42 (s, 3H), 2.34 (m, 4H), 2.19 (s,3H), 1.67 (d, J=6.0 Hz, 3H); ESI-MS: calcd for (C25H29FN8O) 476, found477 (MH⁺).

Example 121

From compound 118, with the same procedure as described in example 120,compound 121 was prepared. ¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (br, 1H),9.38 (br, 1H), 7.23 (d, J=8.8 Hz, 1H), 6.92 (t, J=8.0 Hz, 1H), 6.28 (s,1H), 5.80-5.60 (m, 4H), 3.70 (s, 3H), 3.60 (br, 4H), 3.40 (m, 4H), 2.42(s, 3H), 1.67 (d, J=6.0 Hz, 3H); ESI-MS: calcd for (C24H26FN7O2) 463,found 464 (MH⁺).

Example 122

From compound 119, with the same procedure as described in example 120,compound 122 was prepared. 1H NMR (400 MHz, DMSO-d₆) δ 11.92 (br, 1H),9.31 (br, 1H), 7.30 (d, J=8.8 Hz, 1H), 6.92 (t, J=8.0 Hz, 1H), 6.28 (s,1H), 6.00 (br, 1H), 5.15 (br, 1H), 3.78 (s, 3H), 3.70 (br, 4H), 2.80 (m,4H), 2.57-2.42 (s, s, 6H), 1.80 (br, 1H), 0.85 (br, 2H), 0.00 (br, 2H);ESI-MS: calcd for (C25H29FN8O) 476, found 477 (MH⁺).

Example 123

From compound 119, with the same procedure as described in example 120,compound 123 was prepared. ¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (br, 1H),9.31 (br, 1H), 7.22 (d, J=8.8 Hz, 1H), 6.92 (t, J=8.0 Hz, 1H), 6.27 (s,1H), 5.90 (br, 1H), 5.15 (br, 1H), 3.68 (s, 3H), 3.60 (br, 4H), 3.40 (m,4H), 2.39 (s, 3H), 1.60 (br, 1H), 0.85 (br, 2H), 0.00 (br, 2H); ESI-MS:called for C24H26FN7O2) 463, found 464 (MH⁺).

Example 124

To a solution of5-((4,6-dichloropyrimidin-2-yl)oxy)-4-fluoro-2-methyl-1H-indole (0.109g, 0.349 mmol), 5-cyclopropyl-N-methyl-1H-pyrazol-3-amine (0.088 g,0.641 mmol), and diisopropylethylamine (0.124 g, 0.961 mmol) inanhydrous dimethylformamide (9 mL) under argon atmosphere was addedsodium iodide (0.106 g, 0.705 mmol). The mixtures were heated to 85° C.overnight (16 h). After cooling, the solvent was removed by vacuum andthen redissolved in dichloromethane/isopropanol (8:2) mixtures (50 mL)and washed with sat. NaHCO₃. The combined organic layers were dried overanhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified byflash chromatography over silica gel with CH₂Cl₂:MeOH (95:5) to givecompound 124 (0.110 g, 77%) as a light yellow solid. ¹H NMR (400 MHz,DMSO-d): δ 12.37 (bs, 1H), 11.27 (s, 1H), 7.93 (s, 2H), 7.09 (d, 1H,J=8.4 Hz), 6.89 (m, 1H), 6.68 (s, 1H), 6.21 (s, 1H), 5.77 (bs, 1H), 3.24(s, 3H), 2.37 (s, 3H), 1.67 (m, 1H), 0.83 (m, 2H), 0.51 (m, 2H). MS(ESI): Calcd. for C₂₀H₁₈ClFN₆O: 412, found 413 (M+H).

Example 125

To a solution of6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-N-methylpyrimidin-4-amine(0.105 g, 0.255 mmol), 1-methylpiperazine (0.102 g, 1.021 mmol), andDIPEA (0.099 g, 0.765 mmol) in isopropanol (1.0 mL) was heated to 80° C.for 2 days in a sealed tube. The cooled mixtures were extracted withdichloromethane/isopropanol (8:2) and washed with sat. NaHCO₃. Thecombined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified by flash chromatographyover silica gel with CH₂Cl₂:MeOH (95:5) to give compound 125 (0.083 g,68%) as an off-white solid. ¹H NMR (400 MHz, DMSO-d): δ 11.62 (s, 1H),10.81 (s, 1H), 6.70 (d, 1H, J=8.4 Hz), 6.47 (m, 1H), 5.81 (s, 1H), 5.499s, 1H), 5.25 9s, 1H), 3.04 (m, 4H), 2.84 (s, 3H), 2.00 (s, 3H), 1.93(m, 4H), 1.81 (s, 3H), 1.16 (m, 1H), 0.38 (m, 2H), 0.006 (m, 2H). MS(ESI): Calcd. for C₂₅H₂₉FN₈O: 476, found 478 (M+H).

Example 126

To a solution of5-((4,6-dichloropyrimidin-2-yl)oxy)-4-fluoro-2-methyl-1H-indole (0.109g, 0.349 mmol), 5-cyclopropyl-1-methyl-1H-pyrazol-3-amine (0.088 g,0.641 mmol), and diisopropylethylamine (0.124 g, 0.961 mmol) inanhydrous dimethylformamide (9 mL) under argon atmosphere was addedsodium iodide (0.106 g, 0.705 mmol). The mixtures were heated to 85° C.overnight (16 h). After cooling, the solvent was removed by vacuum andthen redissolved in dichloromethane/isopropanol (8:2) mixtures (50 mL)and washed with sat. NaHCO₃. The combined organic layers were dried overanhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified byflash chromatography over silica gel with CH₂Cl₂:MeOH (9:1) to givecompound 126 (0.255 g, 97%) as an off-white solid. ¹H NMR (400 MHz,DMSO-d): δ 11.32 (s, 1H), 10.31 (bs, 1H), 7.12 (d, 1H, J=8.4 Hz), 6.89(m, 1H), 6.48 (bs, 1H), 6.20 (s, 1H), 5.13 (bs, 1H), 3.59 (bs, 3H), 2.39(s, 3H), 1.53 (m, 1H), 0.61 (m, 2H),−0.26 (m, 2H). MS (ESI): Calcd. forC₂₀H₁₈ClFN₆O: 412, found 413 (M+H).

Example 127

To a solution of6-chloro-N-(5-cyclopropyl-1-methyl-1H-pyrazol-3-yl)-2-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)pyrimidin-4-amine(0.100 g, 0.242 mmol), 1-methylpiperazine (0.097 g, 0.969 mmol), andDIPEA (0.094 g, 0.727 mmol) in isopropanol (1.0 mL) was heated to 80° C.for 2 days in a sealed tube. The cooled mixtures were extracted withdichloromethane/isopropanol (8:2) and washed with sat. NaHCO₃. Thecombined organic layers were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified by flash chromatographyover silica gel with CH₂Cl₂:MeOH (95:5) to give compound 127 (0.106 g,92%) as a light yellow solid. ¹H NMR (400 MHz, DMSO-d): δ 11.32 (s, 1H),9.35 (s, 1H), 7.18 (d, 1H, J=8.4 Hz), 6.93 (m, 1H), 6.28 (s, 1H), 5.98(bs, 1H), 5.27 (s, 1H), 3.68 (s, 1H), 3.52 (m, 4H), 3.26 (dd, 3H, J=5.2,0.4 Hz), 2.49 (s, 3H), 2.44 (m, 4H0, 2.30 (s, 3H), 1.64 (m, 1H), 0.72(m, 2H), 0.00 (m, 2H). MS (ESI): Calcd. for C₂₅H₂₉FN₈O: 476, found 478(M+H).

The invention further encompasses pharmaceutical compositions comprisingany one or more of the compounds disclosed herein and said compounds inthe form of pharmaceutically acceptable salts, hydrates, solvates,crystal forms and individual steroisomers thereof (e.g, diastereomers,enantamers), and as in a compostion with a pharmaceutically acceptablecarrier. These include, but are not limited to, wherein the inventivecompounds are formulated into a composition in a neutral or salt form.

“Pharmaceutically acceptable salts” include the acid addition salts(formed with the free amino groups of the protein) which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such as organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed with the free carboxyl groups also can be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, and procaine and the like.

“Salts” are chemical combinations of two ionizable components (e.g.,when dissolved in water), one acidic and the other basic with respect toone another. If in a salt form, a drug can be either the acidic or thebasic component.

“Pharmaceutically acceptable salts” include any salt form of thecompound wherein the salt is safe for animal ingestion (e.g., nontoxicto humans when taken orally). Exemplary such salts that can be used inaccordance with the invention include, but are not limited to,2-hydroxyethanesulfonate, 2-naphthalenesulfonate,3-hydroxy-2-naphthoate, 3-phenylpropionate, acetate, adipate, alginate,amsonate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate,bisulfate, bitartrate, borate, butyrate, calcium edetate, camphorate,camphorsulfonate, camsylate, carbonate, citrate, clavulariate,cyclopentanepropionate, digluconate, dodecylsulfate, edetate, edisylate,estolate, esylate, ethanesulfonate, finnarate, gluceptate,glucoheptanoate, gluconate, glutamate, glycerophosphate,glycollylarsanilate, hemisulfate, heptanoate, hexafluorophosphate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, laurylsulphonate, malate, maleate, mandelate,mesylate, methanesulfonate, methylbromide, methylnitrate, methylsulfate,mucate, naphthylate, napsylate, nicotinate, nitrate, N-methylglucamineammonium salt, oleate, oxalate, palmitate, pamoate, pantothenate,pectinate, persulfate, phosphate, phosphateldiphosphate, picrate,pivalate, polygalacturonate, propionate, p-toluenesulfonate, saccharate,salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate,suramate, tannate, tartrate, teoclate, thiocyanate, tosylate,triethiodide, undecanoate, and valerate salts, and the like (see also S.M. Berge et al., Pharmaceutical Salts, J. Pharm. Scis., 1977, 66:1-18;P. L. Gould, Salt selection for basic drugs, Int'l J. Pharms. 1986,33:201-17.)

“Solvates” are compositions which, during the process of crystallizationof a compound from solution, trap molecues of the solvent in the forminglattice.

“Hydrates” are solvates wherein the solvent was watter.

“Crystal” forms are solid compostions wherein the molecules making upthe compostion are packed in a repeating lattice structure. When morethan one lattice pattern is possible for compostions made up the samemolecules, the different compositions are called “polymorphs.”

“Diastereomers” are stereoisomers that are not related as object andmirror image, but still differ is in the arrangement inthree-dimensional space about one tetrahedral, sp3-hybridized carbon.

An “enantiomer” is one of two stereoisomers that are mirror images ofeach other, but are non-superposable (not identical).

“Pharmaceutically acceptable carrier” is any excipient which isnon-toxic and aids in a drug's function (see also, Rowe R C et al.,Handbook of Pharmaceutical Excipients, 5^(th) ed., 2006.)

Example 128

This example tests the inhibitory properties of representative compoundsof the invention in c-Src kinase, Aurora-A kinase, Flt3 kinase, Retkinase and TrkA Kinase assays (see, Daniele Fancelli et al, J. Med.Chem., 2006, 49 (24), pp 7247-7251). The KinaseProfilerr™ Service AssayProtocols (Millipore) was used to test the kinase inhibiting activity ofnovel compounds from this invention. To do this, the buffer compositionwas as: 20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1%β-mercaptoethanol, 1 mg/mL BSA. Test compounds were initially dissolvedin DMSO at the desired concentration, then serially diluted to thekinase assay buffer. In a final reaction volume of 25 μL, Aurora-A(h)(5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μMLRRASLG (Kemptide), 10 mM MgAcetate and [γ³³P-ATP]. The reaction wasinitiated by the addition of the MgATP mix. After incubation for 40minute at room temperature, the reaction was stopped by addition of 5 μLof a 3% phosphoric acid solution. 10 μL of the reaction was then spottedonto a P30 filtenat and washed three times for 5 minutes in 50 mMphosphoric acid and once in methanol prior to drying and scintillationcounting. Wells containing substrate but no kinase and wells containinga phosphopeptide control were used to set 0% and 100% phosphorylationvalue, respectively.

The Kinase Hotspot℠ kinase assay was also used to test the compounds forIC50 or % inhibitions (Reaction Biology Corp.). Inhibitory IC50 valueswere determined by titration of compound at the optimal kinaseconcentration (Kinase EC50).

Table 1 shows representative data for the inhibition of abl kinase,Aurora-A kinase, c-Src kinase, Flt3 kinase, KDR kinase and Ret Kinase bythe compounds of this invention at a concentration of 1 μM.

TABLE 1 % Inhibition @1 μM Example No. Abl Auroro-A cSrc Flt3 KDR Ret 10112 99 100 100 95 101 11 111 100 101 100 96 101 12 112 100 100 100 96101 13 112 100 100 101 96 100 15 108 101 99 100 91 101 16 103 101 100 9892 101 17 108 100 98 100 93 98 18 107 100 99 100 93 97 19 108 100 99 9993 100 20 107 99 99 100 93 100 21 107 100 98 101 87 100 22 108 101 100100 92 101 23 108 101 100 100 93 100 25 108 101 100 100 93 101 26 109101 100 100 93 101 27 108 101 99 100 92 101 28 108 101 96 100 89 101 30110 100 99 100 94 101 31 115 101 104 101 91 102 38 108 80 98 98 95 10139 110 98 97 98 95 101 40 110 100 99 98 95 101 41 108 87 97 99 94 101 42110 93 101 100 96 102 43 111 87 101 99 95 102 44 94 97 102 100 90 100 45107 99 107 101 95 102 53 109 97 98 99 95 98 54 108 85 99 99 96 100 55110 101 101 99 96 101 56 111 99 100 99 96 101 57 111 96 99 100 95 102 58112 90 101 99 96 102 59 112 94 100 99 96 102 60 111 97 101 99 95 102 61112 95 100 100 97 102 62 111 102 101 99 96 102 67 112 99 99 100 91 10068 107 99 96 100 92 99 74 111 99 100 100 96 102 81 110 99 99 101 96 9982 106 100 100 100 95 100 83 110 100 99 99 96 101 84 103 98 100 100 93100 87 112 99 95 100 95 83 88 113 100 100 100 96 99 89 112 100 100 10096 99 90 112 100 101 100 95 99 92 111 100 100 100 96 102 93 111 102 101100 95 102 94 111 100 100 99 96 102 95 110 100 100 100 96 101 96 110 98101 99 95 102 98 111 98 100 100 96 102 99 113 101 103 102 91 102 103 11397 96 99 91 101 105 112 98 96 99 91 101 106 109 97 98 99 95 98 107 11195 96 98 90 99 112 113 100 104 102 91 102 120 115 101 104 101 91 101 121116 91 104 102 92 102 122 109 101 104 102 91 101 123 114 100 104 101 88102

These results demonstrate that numerous embodiments of the inventionwith have excellent kinase inhibitory properties for a wide range ofkinases.

Example 129

The embodiment of the invention disclosed in Example 81 (also known ascompound “NTW-3475”) demonstrated strong inhibition of all the kinasesin Example 128 and was chosen for further testing.

The NCI-60 DTP Human Tumor Cell Line Panel was used to further evaluatethe biochemistry of NTW-3475 (see Shoemaker: The NCI60 human tumour cellline anticancer drug screen, Nature Reviews Cancer 6, 813-823 (1 Oct.2006)).

The effect of NTW-3475 on kinase activity was tested for a wide range ofkinases and is shown in Table 2:

Summary Sheet, Activity %

ATP Concentration: Km

Km NTW-3475@ 0.1 μM Abl(h) −9 Abl(m) −2 Abl (H396P) (h) −3 Abl(M351T)(h) −2 Abl (Q252H) (h) −2 Abl(T315l)(h) −2 Abl(Y253F)(h) −2 ACK1(h) 4 ALK(h) 17 ALK4(h) 28 Arg(h) −3 AMPKα1(h) 17 AMPKα2(h) 5 Arg(m) −2ARK5(h) 2 ASK1(h) 112 Aurora-A(h) 7 Aurora-B(h) 1 Aurora-C(h) 21 Axl(h)34 Blk(h) 9 Blk(m) 14 Bmx(h) 1 BRK(h) 33 BrSK1(h) 53 BrSK2(h) 44 BTK(h)37 BTK(R28H)(h) 103 CaMKI(h) 90 CaMKIIβ(h) 99 CaMKIIγ(h) 87 CaMKIδ(h) 87CaMKIIδ(h) 98 CaMKIV(h) 106 CDK1/cyclinB(h) 81 CDK2/cyclinA(h) 97CDK2/cyclinE(h) 89 CDK3/cyclinE(h) 59 CDK5/p25(h) 60 CDK5/p35(h) 77CDK6/cyclinD3(h) 65 CDK7/cyclinH/MAT1 (h) 44 CDK9/cyclin T1(h) 52CHK1(h) 113 CHK2(h) 95 CHK2(II57T)(h) 112 CHK2(Rl45W)(h) 91 CK1γ1(h) 96CK1γ2(h) 134 CK1γ3(h) 117 CK1δ(h) 91 CK1(y) 100 CK2(h) 85 CK2α2(h) 115CLK2(h) 90 CLK3(h) 87 cKit(h) 84 cKit(D816V)(h) 63 cKit(D816H)(h) 9cKit(V560G)(h) 3 cKit(V654A)(h) 32 CSK(h) 75 c-RAF(h) 94 cSRC(h) 2DAPK1(h) 88 DAPK2(h) 106 DCAMKL2(h) 87 DDR2(h) 9 DMPK(h) 102 DRAK1(h) 51DYRK2(h) 96 eEF-2K(h) 86 EGFR(h) 108 EGFR(L858R)(h) 72 EGFR(L861Q)(h) 52EGFR(T790M)(h) 61 EGFR(T790M, L858R)(h) 17 EphA1(h) 12 EphA2(h) −1EphA3(h) 22 EphA4(h) 23 EphA5(h) I22 EphA7(h) 54 EphA8(h) 16 EphB2(h) 21EphB1(h) −1 EphB3(h) 99 EphB4(h) 14 ErbB4(h) 90 FAK(h) 78 Fer(h) 69Fes(h) 32 FGFR1(h) −1 FGFR1(V561M)(h) −4 FGFR2(h) 0 FGFR2(N549H)(h) −1FGFR3(h) 0 FGFR4(h) 19 Fgr(h) 2 Flt1(h) 11 Flt3(D835Y)(h) 0 Flt3(h) 1Flt4(h) 0 Fms(h) 8 Fms(Y969C)(h) 4 Fyn(h) 1 GCK(h) 13 GRK5(h) 100GRK6(h) 69 GRK7(h) 97 GSK3α(h) 96 GSK3β(h) 106 Haspin(h) 96 Hck(h) 18Hck(h) activated 12 HIPK1(h) 91 HIPK2(h) 101 HIPK3(h) 88 IGF-1R(h) 5IGF-1R(h), activated 18 IKKα(h) 102 IKKβ(h) 109 IR(h) 30 IR(h),activated 13 IRR(h) 6 IRAK1(h) 96 IRAK4(h) 67 ltk(h) 29 JAK2(h) 8JAK3(h) 3 JNK1α1(h) 92 JNK2α2(h) 107 JNK3(h) 111 KDR(h) 14 Lck(h) −8Lck(h) activated −1 LIMK1(h) 15 LKB1(h) 98 LOK(h) 20 Lyn(h) 5 Lyn(m) 1MAPK1(h) 128 MAPK2(h) 105 MAPK2(m) 101 MAPKAP-K2(h) 105 MAPKAP-K3(h) 103MEK1(h) 63 MARK1(h) 28 MELK(h) 40 Mer(h) 2 Met(h) 11 Met(D1246H)(h) 18Met(D1246N)(h) 20 Met(M1268T)(h) 22 Met(Yl248C)(h) 20 Met(Yl248D)(h) 21Met(Yl248H)(h) 16 MINK(h) 75 MKK4(m) 100 MKK6(h) 113 MKK7β(h) 97 MLCK(h)110 MLKl(h) 7 Mnk2(h) 92 MRCKα(h) 102 MRCKβ(h) 108 MSK1(h) 104 MSK2(h)90 MSSK1(h) 110 MST1(h) 24 MST2(h) 79 MST3(h) 9 mTOR(h) 115mTOR/FKBP12(h) 63 MuSK(h) 48 NEK2(h) 101 NEK3(h) 82 NEK6(h) 90 NEK7(h)98 NEK11(h) 102 NLK(h) 96 p70S6K(h) 84 PAK2(h) 65 PAK4(h) 70 PAK5(h) 29PAK6(h) 85 PAR-1Bα(h) 15 PASK(h) 91 PEK(h) 103 PDGFRα(h) 67PDGFRα(D842V)(h) 17 PDGFRα(V561D)(h) 8 PDGFRβ(h) 89 PDK1(h) 103 PhKγ2(h)77 Pim-1(h) 114 Pim-2(h) 96 Pim-3(h) 99 PKA(h) 111 PKBα(h) 109 PKBβ(h)97 PKBγ(h) 106 PKCγ(h) 95 PKCβ1(h) 92 PKCβII(h) 109 PKCγ(h) 102 PKCδ(h)90 PKC

(h) 99 PKCη(h) 101 PKCι(h) 105 PKCμ(h) 95 PKC⊖(h) 109 PKCζ(h) 109PKD2(h) 94 PKGlα(h) 100 PKGIβ(h) 111 Plk1(h) 107 Plk3(h) 95 PRAK(h) 100PRK2(h) 30 PrKX(h) 68 PTK5(h) 17 Pyk2(h) 59 Ret(h) −1 Ret (V804L)(h) −4Ret(V804M)(h) 0 RIPK2(h) 90 ROCK-I(h) 93 ROCK-II(h) 58 ROCK-II(r) 42Ron(h) 34 Ros(h) 46 Rse(h) 55 Rsk1(h) 84 Rsk1(r) 69 Rsk2(h) 65 Rsk3(h)25 Rsk4(h) 66 SAPK2a(h) 79 SAPK2a(T106M)(h) 95 SAPK2b(h) 103 SAPK3(h)115 SAPK4(h) 91 SGK(h) 107 SGK2(h) 107 SGK3(h) 102 SIK(h) 33 Snk(h) 97Src(1-530)(h) −2 Src(T341M)(h) 1 SRPK1(h) 96 SRPK2(h) 99 STK33(h) 101Syk(h) 84 TAK1(h) 65 TAO1(h) 65 TAO2(h) 6 TAO3(h) 16 TBK1(h) 28 Tec(h)activated 52 TGFBR1(h) 44 Tie2 (h) 0 Tie2(R849W)(h) 6 Tie2(Y897S)(h) 3TLK2(h) 5 TrkA(h) 2 TrkB(h) 1 TSSK1(h) 86 TSSK2(h) 84 Txk(h) 20 ULK2(h)46 ULK3(h) 11 WNK2(h) 99 WNK3(h) 86 VRK2(h) 99 Yes(h) 0 ZAP-70(h) 110ZIPK(h) 104

NTW-3475 showed strong kinase inhibition for a wide range of kinases(Table 2 and FIGS. 1-2), including mutant kinases wherein the mutationsthought to be critical for their transformation of the cancer cells theyisolated from were tested and, in particular, mutant abl kinases forwhich no inhibitors were known (FIG. 2).

Example 130

NTW-3475 was also tested for its antiproliferative potential using celllines from the NCI 60 cancer cell line panel.

The human tumor cell lines of the cancer screening panel are grown inRPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.For a typical screening experiment, cells are inoculated into 96 wellmicrotiter plates in 100 μL at plating densities ranging from 5,000 to40,000 cells/well depending on the doubling time of individual celllines. After cell inoculation, the microtiter plates are incubated at37° C., 5% CO2, 95% air and 100% relative humidity for 24 h prior toaddition of experimental drugs.

After 24 h, two plates of each cell line are fixed in situ with TCA, torepresent a measurement of the cell population for each cell line at thetime of drug addition (Tz). Experimental drugs are solubilized indimethyl sulfoxide at 400-fold the desired final maximum testconcentration and stored frozen prior to use. At the time of drugaddition, an aliquot of frozen concentrate is thawed and diluted totwice the desired final maximum test concentration with complete mediumcontaining 50 μg/ml gentamicin. Additional four, 10-fold or ½ log serialdilutions are made to provide a total of five drug concentrations pluscontrol. Aliquots of 100 μl of these different drug dilutions are addedto the appropriate microtiter wells already containing 100 μl of medium,resulting in the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48 hat 37° C., 5% CO2, 95% air, and 100% relative humidity. For adherentcells, the assay is terminated by the addition of cold TCA. Cells arefixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA(final concentration, 10% TCA) and incubated for 60 minutes at 4° C. Thesupernatant is discarded, and the plates are washed five times with tapwater and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4%(w/v) in 1% acetic acid is added to each well, and plates are incubatedfor 10 minutes at room temperature. After staining, unbound dye isremoved by washing five times with 1% acetic acid and the plates are airdried. Bound stain is subsequently solubilized with 10 mM trizma base,and the absorbance is read on an automated plate reader at a wavelengthof 515 nm. For suspension cells, the methodology is the same except thatthe assay is terminated by fixing settled cells at the bottom of thewells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA).Using the seven absorbance measurements [time zero, (Tz), controlgrowth, (C), and test growth in the presence of drug at the fiveconcentration levels (Ti)], the percentage growth is calculated at eachof the drug concentrations levels. Percentage growth inhibition iscalculated as:[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz.

Three dose response parameters are calculated for each experimentalagent. Growth inhibition of 50% (C150) is calculated from[(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation.

As shown in FIG. 3 NTW-3475 demonstrated strong antiproliferativeactivity in at least 13 of the 16 cell lines studied. Thisantiproliferative effect was seen a range of cell lines including celllines from Chronic Myelogenous Leukemia, Acute Myelogenous Leukemia,Thyroid, Endometrial, Gastric, Breast and Pancreatic Carcinomas.

Example 131

This example assesses NTW-3475's anti-tumor activity using a scid mousexenograft models for leukemias and nude mouse xenograft models ofendometrial, pancreatic and thyroid carcinomas. NTW-3475 combinationtherapy was also studied in murine xenografts.

The objective of the first study was to evaluate antitumor activities ofthe novel multiple-kinase inhibitor NTW-3475 against human MV411 humanacute myelogenous leukemia (AML) xenograft model in female SCID mice.

Four or six animals were randomly assigned to each study group. Eachanimal was injected subcutaneously in the left and right flank area with0.1 ml of 1.0×10⁸ MV411 cells per mL.

Each test or control animal was placed on a 5-day on, two-day off fortwo cycles of vehicle negative control or 25, 50 mg/kg of NTW-3475.Tumor growth was measured with a digital hand held caliper twice weekly(once tumor emerges) prior to the first dosing and then two to threetimes weekly until euthanized. Animals were weighed prior to tumor cellinjection, prior to dosing, two to three times weekly with tumor growthmeasurements, and prior to euthanasia.

The results are shown in FIG. 4 (tumor weight over time), 5 (tumor sizeover time), and 6 (T/C at day 22). The results indicate that NTW-3475has a strong anti-tumor effect against AML with minimal body weightlost.

The objective of the second study was to evaluate antitumor activitiesof novel multiple-kinase inhibitor NTW-3475 in human K562 chronicmyelogenous leukemia (CML) xenograft model in female SCID mice.

Four to six animals were randomly assigned to study groups. Each animalwas weighed, and then injected subcutaneously in the left and rightflank area with 0.1 ml of 8.0×10⁷ K562 cells per mL.

Each test or control animal was on a 5-day on, two-day off for twocycles treatment schedule. Tumor growth was measured with a digital handheld caliper twice weekly (once tumor emerges) prior to the first dosingand then two to three times weekly until euthanized. Animals wereweighed prior to tumor cell injection, prior to dosing, two to threetimes weekly with tumor growth measurements, and prior to euthanasia.The results are shown in FIGS. 7 and 8. The results show that NTW-3475has a strong anti-tumor effect against CML in this model system.

The objective of the third study was to evaluate antitumor activities ofnovel multiple-kinase inhibitor NTW-3475 in human MIAPaCa-2 pancreaticcarcinoma xenograft in athymic nude-Faxn1 mice.

Four animals were randomly assigned to study groups. Each animal wasweighed, and then injected subcutaneously in the left and right flankarea with 0.1 ml of 5.0×10⁷ MIAPaCa-2 cells per mL.

Each test or control animal was on a 5-day on, two-day off for twocycles treatment schedule with negative control vehicle, positivecontrol Abraxane at 20 mg/kg or 25, 50 mg/kg of NTW-3475. Tumor growthwas measured with a digital hand held caliper twice weekly (once tumoremerges) prior to the first dosing and then two to three times weeklyuntil euthanized. The results are shown in FIGS. 9, 10 and 11 whichindicate that NTW-3475 has a strong anti-tumor effect against pancreaticcarcinoma cells in this model system.

NTW-3475 was similarly tested and shown to have an anti-tumor effect inTT human Thyroid carcinoma xenograft (FIGS. 12 and 13).

NTW-3475 was similarly tested and shown to have an anti-tumor effect inAN3 human endometrial carcinoma carcinoma xenograft (FIGS. 14, 15, and16).

NTW-3475 was similarly tested alone and in combination with Abraxane andthe combination shown to have an anti-tumor effect in MIAPaCa-2xenograft (FIGS. 17, 18 and 19).

Overall the kinase, proliferation, and xenografts studies indicate thatNTW-3475 shows high cellular potency and targeting (FIG. 20) andNTW-3475 is active in vivo against AML, CML, thyroid, endometrial andsome pancreatic carcinoma in xenograft model systems (FIG. 21).

Example 132

The embodiment of the invention disclosed in Example 87 (also known ascompound “NTW-3456”) demonstrated strong inhibition of all the kinasesin Example 128 and was chosen for further testing.

The first study was designed to assess the effect of NTW-3456 on kinaseactivity. Activity of NTW-3456 for a wide range of kinases and is shownFIGS. 22-24, including mutant kinases thought to play a large role inneoplastic transformation (FIGS. 23 and 24). In addition, NTW-3456inhibited kinase activity in abl mutants for which no prior inhibitorshave been FDA approved. In particular, NTW-3456 inhibits the 2 T315Imutant for which no kinase inhibitor had been known prior to NTW-3456.

Example 133

NTW-3456 was tested for its effect on in vitro proliferation using theprotocol for the 60 NCI Cancer Cell Lines used to test NTW-3475.

The NCI-60 DTP Human Tumor Cell Line Panel was used to further evaluatethe biochemistry of NTW-3475 (see Shoemaker: The NCI60 human tumour cellline anticancer drug screen, Nature Reviews Cancer 6, 813-823 (1 Oct.2006)). The effect of NTW-3456 on kinase activity was tested for a widerange of kinases and is shown in FIG. 25. Overall, NTW-3456 inhibitedboth phosphorylation and proliferation. Anti-proliferative activity fromNTW-3456 was seen against AML, CML, thyroid, endometrial, gastric,breast, and some pancreatic carcinoma cell lines (FIG. 26).

Example 134

This example studies the anti-tumor activity of NTW-3456 in xenograftmodel systems for AML, CML, thyroid, endometrial, pancreatic carcinomas.

FIGS. 27-32 depict the results of various concentrations of NTW-3456 ontumor growth in scid mouse/xenograft model of AML using MV411 cells. Inthis model system, NTW-3456 demonstrates significant anti-tumoractivity, especially at higher doses (FIGS. 29 and 32). This inventionis characterized as it is here was a correlation between the inhibitionof the pFLT3 kinase and growth control.

The mice carrying the established MV4-11 tumor xenografts (tumor volumearound 150 mm³) were given an oral dose of 50 mg/kg NTW-3456. At 0, 1,2, 8, 16, 24 hours after dosing, three were euthanized. The bloodsamples were collected and plasma was prepared with centrifuge for LCMSassay. And the tumors were harvested, homogenized in lysis buffer,immunoprecipitated with anti-FLT3 antibody FIG. 33.

The curve (solid dot) is the plot of plasma concentration-time profilesof NTW-3456 after oral administration of 50 mg/kg. The top curve (solidtriangle) showed NTW-3456 inhibit FLT3 phosphorylation (pFLT3) of MV4-11tumors after oral administration of 50 mg/kg NTW-3456 at indicated time.

NTW-3456 inhibits FLT3 phosphorylation in vivo and it is time-dependent.After 24 hour, NTW-3456 (7 nM in plasma) still inhibits more than 60%FLT3 phoseporylation of MV4-11 tumor.

FIGS. 34 and 35 depict the results of various concentrations of NTW-3456on a scid mouse/xenograft model of CML using K562 cells. In this modelsystem, NTW-3456 demonstrates significant anti-tumor activity,especially at higher doses (FIG. 34).

FIGS. 36-38 depict the results of various concentrations of NTW-3456 ona nude mouse/xenograft the TT cell model of thyroid carcinoma. In thismodel system, NTW-3456 demonstrates significant anti-tumor activity,especially at higher doses (FIG. 38).

FIGS. 39-41 depict the results of various concentrations of NTW-3456 onthe AN3 nude mouse/xenograft model of endometrial carcinoma. In thismodel system, NTW-3456 demonstrates significant anti-tumor activity,especially at higher doses (FIG. 41).

FIGS. 42-44 depict the results of various concentrations of NTW-3456 ona nude mouse/xenograft the MIAPaCa-2 model of pancreatic carcinoma. Inthis model system, NTW-3456 demonstrates some anti-tumor activity inthis model of pancreatic carcinoma.

FIGS. 45 and 46 depict the results of various concentrations ofNTW-3456, with or without Abraxane on a MiaPaCa-2 model of pancreaticcarcinoma. In this model system, NTW-3456 demonstrates significantanti-tumor activity, especially when used in combination with Abraxane(FIG. 47).

FIGS. 48 and 49 depict the results of various concentrations ofNTW-3456, with or without Abraxane on a Panc-1 model of pancreaticcarcinoma. In this model system, NTW-3456 demonstrates significantanti-tumor activity, especially when used in combination with Abraxane

FIG. 50 summarizes NTW-3456's anti-tumor activity in murine xenografts.Overall, these examples demonstrate the high specific activity seen withNTW-3456

EXAMPLE 135

This example studies the biochemistry of NTW-3456 and NTW-3475 invarious cells.

FIGS. 51 and 52 show the dose response curves for the inhibition ofproliferation and pERK and pAKt signaling in MiaPaCa-2 and BxPC3 cellsby NTE-3456 and NTW-3475, respectively. FIG. 53 summarizes this data.

FIG. 54 shows the dose response curves for the inhibition of in vitrogrowth in K562 cells by NTE-3456, Nilotinib, Ponatinnib, Suntinib, andImatinib. FIG. 55 shows the dose response curves for the inhibition ofp-Crl kinase activity in K562 cells by NTE-3456, Nilotinib, Ponatinnib,Suntinib, and Imatinib. FIG. 56 shows the dose response curves for theinduction of Caspse 3/7 activity in K562 cells by NTE-3456, Nilotinib,Ponatinnib, Suntinib, and Imatinib. FIG. 57 summarizes this data.

FIG. 58 shows the dose response curve for the inhibition of kinaseactivity in BaF3 cells by NTW-3456 from FGFR1, FGFR2, FGFR3, and FGFR4.FIG. 59 sets forth the NTW-3456 IC50 levels for FGFR1, FGFR2, FGFR3, andFGFR4 driven BaF3 cells. Overall, the inhibition of proliferation, pERKpathway signaling in MIaPaca-2 and BxPC3 cells by NTW-3475 (FIG. 52) andNTW-3456 (FIG. 51) are similar. Further, these results suggest acorrelation between growth inhibition and the signaling inhibition.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of treating cancer comprising a tumorcell, comprising contacting the tumor cell with a composition comprisinga compound of formula (I):

or its pharmaceutically acceptable salts, hydrates, solvates, andindividual diastereomers thereof, wherein R is selected from the groupconsisting of: (i) hydrogen, amino, and alkyl amino; (ii) C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl; (iii) K—Ar, wherein: Ar representsheteroaryl or aryl, each of which is substituted with from 0 to 4substituents independently chosen from the group consisting of: (1)halogen, hydroxy, amino, amide, cyano, —COOH, —SO₂NH₂, oxo, nitro andalkoxycarbonyl; and (2) C₁-C₆ alkyl, C₁-C₆ alkoxy, C₃-C₁₀ cycloalkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₂-C₆ alkanoyl, C₁-C₆ haloalkyl, C₁-C₆haloalkoxy, mono- and di- (C₁-C₆alkyl)amino, C₁-C₆ alkylsulfonyl, mono-and di-(C₁-C₆alkyl) sulfonamido and mono- anddi-(C₁-C₆alkyl)aminocarbonyl; phenylC₀-C₄alkyl and (4- to 7-memberedheterocycle)C₀-C₄ alkyl, each of which is substituted with from 0 to 4secondary substituents independently chosen from halogen, hydroxy,cyano, oxo, imino, C₁-C₄ alkyl, C₁-C₄ alkoxy and C₁-C₄ haloalkyl; K isselected from the group consisting of: 1) O, S, SO, and SO₂; 2)(CH₂)_(m), m=0-3, —O(CH₂)_(p), p=1-3, —S(CH₂)_(p), p=1-3, —N(CH₂)p,p=1-3, and —(CH₂)_(p)O, p=1-3; and 3) NR₁, wherein R₁ is selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,alkylthio, aryl, and arylalkyl; and (iv) groups of the formula (Ia):

 wherein: R₂ is selected from the group consisting of hydrogen, C₁-C₄alkyl, and oxo; and X is CH, when R₃ is hydrogen; or X—R₃ is O; or X isN; R₃ is selected from the group consisting of hydrogen, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀ aryl and heteroaryl,(C₃-C₇cycloalkyl)C₁-C₄ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆alkylthio, C₂-C₆ alkanoyl, C₁-C₆ alkoxycarbonyl, C₂-C₆ alkanoyloxy,mono- and di-(C₃-C₈ cycloalkyl)amino C₀-C₄ alkyl, (4- to 7-memberedheterocycle)C₀-C₄alkyl, C₁-C₆ alkylsulfonyl, mono- and di-(C₁-C₆ alkyl)sulfonamido, and mono- and di-(C₁-C₆alkyl)aminocarbonyl, each of whichis substituted with from 0 to 4 substituents independently chosen fromhalogen, hydroxy, cyano, amino,—COOH or oxo; and Het is selected fromany heterocycle, which is substituted with from 0 to 4 substituentsindependently chosen from the group consisting of: (i) C₁C₆ alkyl, C₂C₆alkenyl, and C₂C₆ alkynyl; (ii) halogen, hydroxy, amino, amide, cyano,—COOH, —SO₂NH₂, oxo, nitro and alkoxycarbonyl; and (iii) aryl; R₁₁ andR₁₂ are independently selected from the group consisting of Hydrogen, F,Cl, Br, CN, C₁-C₄ alkyl, and C₁-C₆ alkoxy; and R₁₃, R₁₄ and R₁₅ areindependently selected from the group consisting of Hydrogen, C₁ -C₄alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀ aryl or heteroaryl, C₁-C₆alkoxy, C₁-C₆ alkylthio, C₂-C₆ alkanoyl, C₁-C₆ alkoxycarbonyl, and C₂-C₆alkanoyloxy.
 2. The method of claim 1, in which the concentration of thecompound in the pharmaceutical composition results in administration toa patient of a dosage of between 0.01-100 mg/kg body weight/day.
 3. Themethod of claim 1, wherein the cancer is breast cancer, gastric cancer,chronic myelogenous leukemia, acute myelogenous leukemia, pancreaticcarcinoma, thyroid cancer or endometrial carcinoma.
 4. A method of claim3, in which the cancer is chronic myeogenous leukemia (CML).
 5. A methodof treating cancer comprising a tumor cell, comprising contacting thetumor cell with a composition comprising a compound having the formula:

or its pharmaceutically acceptable salts, solvates, and individualdiastereomers thereof.
 6. The method of claim 5, in which theconcentration of the compound in the pharmaceutical composition resultsin administration to a patient of a dosage of between 0.01-100 mg/kgbody weight/day.
 7. The method of claim 5, wherein the cancer is breastcancer, gastric cancer, chronic myelogenous leukemia, acute myelogenousleukemia, pancreatic carcinoma, thyroid cancer or endometrial carcinoma.8. A method of claim 7, in which the cancer is chronic myeogenousleukemia (CML).
 9. A method of treating cancer comprising a tumor cell,comprising contacting the tumor cell with a composition comprising acompound having the formula:

or its pharmaceutically acceptable salts, hydrates, solvates, andindividual diastereomers thereof.
 10. The method of claim 9, in whichthe concentration of the compound in the pharmaceutical compositionresults in administration to a patient of a dosage of between 0.01-100mg/kg weight/day.
 11. The method of claim 9, wherein the cancer isbreast cancer, gastric cancer, chronic myelogenous leukemia, acutemyelogenous leukemia, pancreatic carcinoma, thyroid cancer orendometrial carcinoma.
 12. A method of claim 11, in which the cancer ischronic myeogenous leukemia (CML).
 13. A method of claim 11, in whichthe cancer is pancreatic carcinoma.
 14. A method of claim 7, in whichthe cancer is pancreatic carcinoma.